Biocomposites and hybrid biomaterials based on calcium orthophosphates

Abstract

The state-of-the-art of biocomposites and hybrid biomaterials based on calcium orthophosphates that are suitable for biomedical applications is presented in this review. Since these types of biomaterials offer many significant and exciting possibilities for hard tissue regeneration, this subject belongs to a rapidly expanding area of biomedical research. Through successful combinations of the desired properties of matrix materials with those of fillers (in such systems, calcium orthophosphates might play either role), innovative bone graft biomaterials can be designed. Various types of biocomposites and hybrid biomaterials based on calcium orthophosphates, either those already in use or being investigated for biomedical applications, are extensively discussed. Many different formulations, in terms of the material constituents, fabrication technologies, structural and bioactive properties as well as both in vitro and in vivo characteristics, have already been proposed. Among the others, the nanostructurally controlled biocomposites, those containing nanodimensional compounds, biomimetically fabricated formulations with collagen, chitin and/or gelatin as well as various functionally graded structures seem to be the most promising candidates for clinical applications. The specific advantages of using biocomposites and hybrid biomaterials based on calcium orthophosphates in the selected applications are highlighted. As the way from the laboratory to the hospital is a long one, and the prospective biomedical candidates have to meet many different necessities, this review also examines the critical issues and scientific challenges that require further research and development.

Introduction

The fracture of bones due to various traumas or natural aging is a typical type of a tissue failure. An operative treatment frequently requires implantation of a temporary or a permanent prosthesis, which is still a challenge for orthopedic surgeons, especially in the case of large bone defects. A rapidly aging population and serious drawbacks to using natural bone grafts make the situation even worse; therefore, there is a high clinical demand for bone substitutes. Unfortunately, medical application of xenografts (e.g., bovine bone) is generally associated with potential viral infections. In addition, xenografts have a low osteogenicity, an increased immunogenicity and usually resorb more rapidly than autogenous bone. Similar limitations are also valid for human allografts (i.e., tissue transplantation between individuals of the same species but of nonidentical genetic composition), where the concerns about potential risks of transmitting tumor cells, a variety of bacterial and viral infections as well as immunological and blood group incompatibility are even stronger.^1-3 Moreover, harvesting and conservation of allografts (exogenous bones) are additional limiting factors. Autografts (endogenous bones) are still the “golden standard” among any substitution materials, because they are osteogenic, osteoinductive, osteoconductive, completely biocompatible, non-toxic and do not cause any immunological problems (non-allergenic). They contain viable osteogenic cells, bone matrix proteins and support bone growth. Usually, autografts are well accepted by the body and rapidly integrate into the surrounding bone tissues. For these reasons, they are routinely used for a long period with good clinical results,^3-6 and it is fair to say that complications mostly arose in the past.^7,8 Unfortunately, a limited number of donor sites restrict the quantity of autografts harvested from the iliac crest or other locations of the patient’s own body. In addition, their medical application always involves additional traumas and scars resulting from the extraction of a donor tissue during a superfluous surgical operation, which requires further healing at the donation site and can involve long-term postoperative pain.^1,8-11 Thus, any types of biologically derived transplants appear to be imperfect solutions, mainly due to a restricted quantity of donor tissues, donor site morbidity as well as potential risks of an immunological incompatibility and disease transfer.^9,11,12 In this light, man-made materials (alloplastic or synthetic bone grafts) stand out as a reasonable option, because they are easily available and might be processed and modified to suit the specific needs of a given application.^13-15 What’s more, there are no concerns about potential infections, immunological incompatibility, sterility or donor site morbidity. Therefore, investigations on artificial materials for bone tissue repair appear to be one of the key subjects in the field of biomaterials research for clinical applications.¹⁶

Currently, there are several classes of synthetic bone grafting biomaterials for in vivo applications.^17-21 Examples include natural coral, coral-derived materials, bovine porous demineralized bone, human demineralized bone matrix, bioactive glasses, glass-ceramics and calcium orthophosphates.¹¹ All of these biomaterials are biocompatible and osteoconductive, guiding bone tissue from the edges toward the center of the defect, and aim to provide a scaffold of interconnected pores, with pore dimensions ranging from 200 µm^22,23 to 2 mm,²⁴ to facilitate tissue and vessel ingrowths. Among them, porous bioceramics made of calcium orthophosphates appear very promising due to both excellent biocompatibility and their ability to bond to living bone in the body. This is directly related to the fact that the inorganic material of mammalian calcified tissues, i.e., of bones and teeth, consists of calcium orthophosphates.^25-27 For this reason, other artificial materials are normally encapsulated by fibrous tissue when implanted in body defects, while calcium orthophosphates are not.²⁸ Many types of calcium orthophosphate-based bioceramics with different chemical composition are already on the market. Unfortunately, as with any ceramic material, calcium orthophosphate bioceramics alone lack the mechanical and elastic properties of calcified tissues. Namely, scaffolds made of calcium orthophosphates suffer from a low elasticity, a high brittleness, a poor tensile strength, a low mechanical reliability and fracture toughness, which leads to various concerns about their mechanical performance after implantation.^29-31 In addition, in many cases, it is difficult to form calcium orthophosphate bioceramics into the desired shapes.

The superior strength and partial elasticity of biological calcified tissues (e.g., bones) are due to the presence of bioorganic polymers (mainly, collagen type I fibers³²) rather than to a natural ceramic (mainly, a poorly crystalline, ion-substituted CDHA, often referred to as “biological apatite”) phase.^34,35 The elastic collagen fibers are aligned along the main stress directions in bone. The biochemical composition of bones is given in Table 1.³⁶ A decalcified bone becomes very flexible and is easily twisted, whereas a bone without collagen is very brittle; thus, the inorganic, nano-sized crystals of biological apatite provide hardness and stiffness, while the bioorganic fibers are responsible for the elasticity and toughness.^26,37 In bones, both types of materials integrate with each other on a nanometric scale in such a way that the crystallite size, fiber orientation, short-range order between the components, etc. determine its nanostructure and, therefore, the function and mechanical properties of the entire composite.^33,38-42 From the mechanical point of view, bone is a tough material at low strain rates but fractures more like a brittle material at high strain rates; generally, it is rather weak in tension and shear, particularly along the longitudinal plane. Besides, bone is an anisotropic material, because its properties are directionally dependent.^25,26,31

Table 1. The biochemical composition* of bones.³⁶ .

Inorganic phases	wt. %	Bioorganic phases	wt. %
calcium orthophosphates (biological apatite)	~60	collagen type I	~20
water	~9	non-collagenous proteins: osteocalcin, osteonectin, osteopontin, thrombospondin, morphogenetic proteins, sialoprotein, serum proteins	~3
carbonates	~4	other traces: polysaccharides, lipids, cytokines	balance
citrates	~0.9	primary bone cells: osteoblasts, osteocytes, osteoclasts	balance
sodium	~0.7
magnesium	~0.5
other traces: Cl-, F-, K+ Sr2+, Pb2+, Zn2+, Cu2+, Fe2+	balance

^* The composition is varied from species to species and from bone to bone.

It remains a great challenge to design the ideal bone graft, one that emulates nature’s own structures or functions. Certainly, the successful design requires an appreciation of the structure of bone. According to expectations, the ideal bone graft should be benign, available in a variety of forms and sizes, all with sufficient mechanical properties for use in load-bearing sites, form a chemical bond at the bone/implant interface as well as be osteogenic, osteoinductive, osteoconductive, biocompatible, completely biodegradable, at the expense of bone growth, and moldable to fill and restore bone defects.^29,40,43 Further, it should resemble the chemical composition of bones (thus, the presence of calcium orthophosphates is mandatory), exhibit contiguous porosity to encourage invasion by the live host tissue as well as possess both viscoelastic and semi-brittle behavior, as bones do.^44-47 Moreover, the degradation kinetics of the ideal implant should be adjusted to the healing rate of the human tissue, with absence of any chemical or biological irritation and/or toxicity caused by substances that are released due to corrosion or degradation. Ideally, the combined mechanical strength of the implant and the ingrowing bone should remain constant throughout the regenerative process. Furthermore, the substitution implant material should not significantly disturb the stress environment of the surrounding living tissue.⁴⁸ Finally, there is the opinion that, in the case of a serious trauma, the bone should fracture rather than the implant.²⁹ A good sterilizability, storability and processability as well as a relatively low cost are also of a great importance to permit clinical application. Unfortunately, no artificial biomaterial is yet available that embodies all these requirements, and it is unlikely that one will appear in the near future. To date, most of the available biomaterials appear to be either predominantly osteogenic or osteoinductive or else purely osteoconductive.²

Careful consideration of the bone type and mechanical properties are needed to design bone substitutes. Indeed, in high load-bearing bones, such as the femur, the stiffness of the implant needs to be adequate: not too stiff to result in strain shielding, but rigid enough to present stability. However, in relatively low load-bearing applications such as cranial bone repairs, it is more important to have stability and the correct three-dimensional shapes for aesthetic reasons. One of the most promising alternatives is to apply materials with similar composition and nanostructure to that of bone tissue.⁴⁰ Mimicking the structure of calcified tissues and addressing the limitations of the individual materials in the development of organic-inorganic hybrid biomaterials provides excellent possibilities for improving conventional bone implants. In this sense, suitable biocomposites tailored to physical, biological and mechanical properties and predictable degradation behavior can be prepared by combining biologically relevant calcium orthophosphates with bioresorbable polymers.^49,50 As a rule, the general behavior of these bioorganic/calcium orthophosphate biocomposites is dependent on nature, structure and relative contents of the constitutive components, although other parameters, such as the preparation conditions, also determine the properties of the final materials. Currently, biocomposites with calcium orthophosphates incorporated as either a filler or a coating (or both) and either into or onto a biodegradable polymer matrix in the form of particles or fibers are increasingly considered for use as bone tissue engineering scaffolds due to their improved physical, biological and mechanical properties.^51-57 In addition, such biocomposites could set out general requirements for the next generation of biomaterials; they should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the implants by the regenerating tissue.^50,58,59 Thus, through the successful combinations of ductile polymer matrixes with hard and bioactive particulate bioceramic fillers, optimal materials can be designed, and, ideally, this approach could lead to a superior construction to be used as either implants or posterior dental restorative material.⁶⁰

A lint-reinforced plaster was the first composite used in clinical orthopedics as an external immobilizer (bandage) in the treatment of bone fracture by Mathijsen in 1852,⁶¹ followed by Dreesman in 1892.⁶² A great progress in the clinical application of various types of composite materials has been achieved since then. Based on past experience and newly gained knowledge, various composite materials with tailored mechanical and biological performance can now be manufactured and used to meet various clinical requirements.⁶³ This review presents only a brief history as well as advances in the field of calcium orthophosphate-based biocomposites and hybrid biomaterials suitable for biomedical application. The majority of the reviewed literature is restricted to the recent publications; a limited number of papers published in the 20th century have been cited. Various aspects of the material constituents, fabrication technologies, structural and bioactive properties as well as phase interactions have been considered and discussed in detail. Finally, several critical issues and scientific challenges that are needed for further advancement are outlined.

General Information on Composites and Biocomposites

According to Wikipedia, the free encyclopedia, “composite materials” (or “composites” for short) are engineered materials made from two or more constituent materials with significantly different physical or chemical properties, which remain separate and distinct on a macroscopic level within the finished structure.”⁶⁴ Thus, composites are always heterogeneous. Furthermore, the phases of any composite retain their identities and properties and are bonded, which is why an interface is maintained between them. This provides improved specific or synergistic characteristics that cannot be obtained by any of the original phases alone.⁶⁵ Following the point of view of some predecessors, we also consider that, “for the purpose of this review, composites are defined as those having a distinct phase distributed through their bulk, as opposed to modular or coated components.”⁶⁶ For this reason, with a few important exceptions, the structures obtained by soaking various materials in supersaturated solutions containing ions of calcium and orthophosphate (reviewed in ref.67–73), those obtained by coating of various materials by calcium orthophosphates (reviewed in ref.74–82) as well as calcium orthophosphates coated by other compounds^83-87 have not been considered; however, composite coatings have been considered. Occasionally, porous calcium orthophosphate scaffolds filled by cells inside the pores^88-91 as well as calcium orthophosphates impregnated by biologically active substances^92,93 are also defined as composites and/or hybrids; nevertheless, such structures have not been considered in this review either.

In any composite, there are two major categories of constituent materials: a matrix (or a continuous phase) and (a) dispersed phase(s). To create a composite, at least one portion of each type is required. General information on the major fabrication and processing techniques may be found elsewhere.^66,94 The continuous phase is responsible for filling the volume as well as surrounding and supporting the dispersed material(s) by maintaining their relative positions. The dispersed phase(s) is(are) usually responsible for enhancing one or more properties of the matrix. Most of the composites target an enhancement of mechanical properties of the matrix, such as stiffness and strength; however, other properties, such as erosion stability, transport properties (electrical or thermal), radiopacity, density or biocompatibility might also be of a great interest. This synergism produces properties that are unavailable from the individual constituent materials.^94,95 What’s more, by controlling the volume fractions and local and global arrangement of the dispersed phase, the properties and design of composites can be varied and tailored to suit the necessary conditions. For example, in the case of ceramics, the dispersed phase serves to impede crack growth. In this case, it acts as reinforcement. A number of methods, including deflecting crack tips, forming bridges across crack faces, absorbing energy during pullout and causing a redistribution of stresses in regions, adjacent to crack tips, can be used to accomplish this.⁹⁶ Other factors to be considered in composites include the volume fraction of (a) dispersed phase(s), its(their) orientation and homogeneity of the overall composite. For example, higher volume fractions of reinforcement phases tend to improve the mechanical properties of the composites, while continuous and aligned fibers best prevent crack propagation, with the added property of anisotropic behavior. Furthermore, the uniform distribution of the dispersed phase is also desirable, as it imparts consistent properties to the composite.^64,94,95

In general, composites might be simple, complex, graded or hierarchical. The term “a simple composite” refers to composites that result from the homogeneous dispersion of one dispersed phase throughout a matrix. The term “a complex composite” refers to composites that result from the homogeneous dispersion of several dispersed phases throughout one matrix. The term “a graded composite” refers to composites that result from the intentionally structurally inhomogeneous dispersion of one or several dispersed phases throughout one matrix. The term “a hierarchical composite” refers to those cases in which fine entities of either a simple or a complex composite are somehow aggregated to form coarser ones (e.g., granules or particles), which afterwards are dispersed inside another matrix to produce the second hierarchical scale of the composite structure. There is another set of four types of composites: (1) fibrous composites, where the fibers are in a matrix; (2) laminar composites, in which the phases are in layers; (3) particulate composites, where the particles or flakes are in a matrix and (4) hybrid composites, which are combinations of any of the above. Yet another classification system of the available composites is based on the matrix materials (metals, ceramics and polymers).⁶³

In most cases, three interdependent factors must be considered in designing of any composite: (1) the selection of a suitable matrix and dispersed materials, (2) the choice of appropriate fabrication and processing methods and (3) both internal and external design of the device itself.⁶⁶ Furthermore, any composite must be formed to shape. To do this, the matrix material can be added before or after the dispersed material has been placed into a mold cavity or onto the mold surface. The matrix material experiences a melding event that, depending upon the nature of the matrix material, can occur in various ways, such as chemical polymerization, setting, curing or solidification from a melted state. Due to a general inhomogeneity, the physical properties of many composite materials are not isotropic, but rather orthotropic (i.e., there are different properties or strengths in different orthogonal directions).^64,94,95

In order to prepare any type of a composite, at least two different materials must be mixed. Thus, a phase miscibility phenomenon appears to be of paramount importance.^97,98 Furthermore, the interfacial strength among the phases is a very important factor, because a lack of adhesion among the phases will result in an early failure at the interface and thus in a decrease in the mechanical properties, especially the tensile strength. From a chemical point of view, we can distinguish several types of interactions among the composite components: materials with strong (covalent, coordination, ionic) interactions; those with weak interactions (van der Waals forces, hydrogen bonds, hydrophilic-hydrophobic balance) and those without chemical interactions among the components.⁹⁹ Wetting is also important in bonding or adherence of the materials. It depends on the hydrophilicity or polarity of the filler(s) and the available polar groups of the matrix.

Biocomposites are defined as non-toxic composites that are able to interact well with the human body in vivo and, ideally, contain one or more component(s) that stimulate(s) the healing process and uptake of the implant.¹⁰⁰ Thus, for biocomposites, biological compatibility appears to be more important than any other type of compatibility.^63,101,102 Interestingly, according to the databases, the first paper with the term “biocomposite” in the title was published in 1987,¹⁰³ and the first one containing a combination of the terms “biocomposite” and HA in the title was published in 1991.¹⁰⁴ Thus, this subject appears to be quite new. The most common properties from the bioorganic and inorganic domains to be combined in biocomposites have been summarized in Table 2.⁴⁰ For general advantages of the modern calcium orthophosphate-based biocomposites over calcium orthophosphate bioceramics and bioresorbable polymers individually, interested readers are advised to see the “Composite Materials Strategy” section of reference 50.

Table 2. General respective properties from the bioorganic and inorganic domains, to be combined in various composites and hybrid materials⁴⁰ .

inorganic	bioorganic
hardness, brittleness	elasticity, plasticity
high density	low density
thermal stability	permeability
hydrophilicity	hydrophobicity
high refractive index	selective complexation
mixed valence slate (red-ox)	chemical reactivity
strength	bioactivity

The Major Constituents of Biocomposites and Hybrid Biomaterials for Bone Grafting

Calcium orthophosphates

The main driving force behind the use of calcium orthophosphates as bone substitute materials is their chemical similarity to the mineral component of mammalian bones and teeth.^25-27 As a result, in addition to being non-toxic, they are biocompatible, not recognized as foreign materials in the body and, most importantly, exhibit both bioactive behavior and the ability to integrate into living tissue by the same processes active in remodeling healthy bone. This leads to an intimate physicochemical bond between the implants and bone, termed osteointegration.¹⁰⁵ More to the point, calcium orthophosphates are also known to support osteoblast adhesion and proliferation.^106,107 Even so, the major limitations to the use of calcium orthophosphates as load-bearing biomaterials are their mechanical properties; namely, they are brittle with poor fatigue resistance.^29-31 Their poor mechanical behavior is even more evident for highly porous ceramics and scaffolds. Because porosity greater than 100 µm is the requirement for proper vascularization and bone cell colonization,^108-110 in biomedical applications, calcium orthophosphates are used primarily as fillers and coatings.²⁷

The complete list of known calcium orthophosphates, including their standard abbreviations and major properties, is given in Table 3, while the detailed information on calcium orthophosphates, their synthesis, structure, chemistry, other properties and biomedical application has been comprehensively reviewed recently in reference.27 Even more thorough information on calcium orthophosphates might be found in special books and monographs.^111-117

Table 3. Existing calcium orthophosphates and their major properties²⁷ .

Ca/P molar ratio	Compound	Formula	Solubility at 25°C, -log(Ks)	Solubility at     25°C, g/L	pH stability range in aqueous solutions at 25°C
0.5	Monocalcium phosphate monohydrate (MCPM)	Ca(H2PO4)2·H2O	1.14	~18	0.0 – 2.0
0.5	Monocalcium phosphate anhydrous (MCPA or MCP)	Ca(H2PO4)2	1.14	~17	[c]
1.0	Dicalcium phosphate dihydrate (DCPD), mineral brushite	CaHPO4·2H2O	6.59	~0.088	2.0 – 6.0
1.0	Dicalcium phosphate anhydrous (DCPA or DCP), mineral monetite	CaHPO4	6.90	~0.048	[c]
1.33	Octacalcium phosphate (OCP)	Ca8(HPO4)2(PO4)4·5H2O	96.6	~0.0081	5.5 – 7.0
1.5	α-Tricalcium phosphate (α-TCP)	α-Ca3(PO4)2	25.5	~0.0025	[a]
1.5	β-Tricalcium phosphate (β-TCP)	β-Ca3(PO4)2	28.9	~0.0005	[a]
1.2 – 2.2	Amorphous calcium phosphates (ACP)	CaxHy(PO4)z·nH2O, n = 3 – 4.5; 15 – 20% H2O	[b]	[b]	~5 – 12 [d]
1.5 – 1.67	Calcium-deficient hydroxyapatite (CDHA or Ca-def HA)[e]	Ca10-x(HPO4)x(PO4)6-x(OH)2-x (0 < x < 1)	~85	~0.0094	6.5 – 9.5
1.67	Hydroxyapatite (HA, HAp or OHAp)	Ca10(PO4)6(OH)2	116.8	~0.0003	9.5 – 12
1.67	Fluorapatite (FA or FAp)	Ca10(PO4)6F2	120.0	~0.0002	7 – 12
1.67	Oxyapatite (OA, OAp or OXA)[f]	Ca10(PO4)6O	~69	~0.087	[a]
2.0	Tetracalcium phosphate (TTCP or TetCP), mineral hilgenstockite	Ca4(PO4)2O	38 – 44	~0.0007	[a]

^[a] These compounds cannot be precipitated from aqueous solutions. ^[b] Cannot be measured precisely. However, the following values were found: 25.7 ± 0.1 (pH = 7.40), 29.9 ± 0.1 (pH = 6.00), 32.7 ± 0.1 (pH = 5.28). The comparative extent of dissolution in acidic buffer is: ACP > > α-TCP > > β-TCP > CDHA > > HA > FA. ^[c] Stable at temperatures above 100°C. ^[d] Always metastable. ^[e] Occasionally, it is called “precipitated HA (PHA).” ^[f] Existence of OA remains questionable.

Polymers

Polymers are a class of materials consisting of large molecules, often containing many thousands of small units, or monomers, joined together chemically to form one giant chain, thus creating very ductile materials. In this respect, polymers are comparable with major functional components of the biological environment: lipids, proteins and polysaccharides. They differ from each other in chemical composition, molecular weight, polydispersity, crystallinity, hydrophobicity, solubility and thermal transitions. Their properties can be fine-tuned over a wide range by varying the type of polymer or chain length as well as by copolymerization or blending of two or more polymers.^118-120 Unlike ceramics, polymers exhibit substantial viscoelastic properties and easily can be fabricated into complex structures, such as sponge-like sheets, gels or complex structures with intricate porous networks and channels.¹²¹ Being X-ray transparent and non-magnetic, polymeric materials are fully compatible with modern diagnostic methods, such as CT and magnetic resonance imaging. Unfortunately, most of them are unable to meet the strict demands of the in vivo physiological environment. Namely, the main requirements for polymers suitable for biomedical applications are that they must be biocompatible, not elicit an excessive or chronic inflammatory response upon implantation and, for those that degrade, they must breakdown into non-toxic products only. Unfortunately, polymers, for the most part, lack rigidity, ductility and, ultimately, the mechanical properties required in load-bearing applications. Thus, despite their good biocompatibility, many of the polymeric materials are mainly used for soft tissue replacements (such as skin, blood vessel, cartilage, ligament replacement, etc.). Moreover, the sterilization processes (autoclave, ethylene oxide and 60 Co irradiation) may affect the polymer properties.¹²²

There are a variety of biocompatible polymers suitable for biomedical applications.^123,124 For example, polyacrylates, poly(acrylonitrile-co-vinylchloride) and polylysine have been investigated for cell encapsulation and immunoisolation.^125,126 Polyorthoesters and PCL have been investigated as drug delivery devices, the latter for long-term sustained release because of its slow degradation rates.¹²⁷ PCL is a hydrolytic polyester with an appropriate resorption period that releases non-toxic byproducts upon degradation.¹²⁸ Other polyesters and PTFE are used for vascular tissue replacement. Polyurethanes are in use as coatings for pacemakers’ lead insulation and have been investigated for reconstruction of the meniscus.^129,130 Polymers considered for orthopedic purposes include polyanhydrides, which have also been investigated as delivery devices (due to their rapid and well-defined surface erosion) and for bone augmentation or replacement, since they can be photopolymerized in situ.^127,131,132 To overcome their poor mechanical properties, they have been copolymerized with imides or formulated to be cross-linkable in situ.¹³² Other polymers, such as polyphosphazenes, can have their properties (e.g., degradation rate) easily modified by varying the nature of their side groups and have been shown to support osteoblast adhesion, which makes them candidate materials for skeletal tissue regeneration.¹³² PPF has emerged as a good bone replacement material, exhibiting good mechanical properties (comparable to trabecular bone), possessing the capability to cross-link in vivo through the C = C bond and being hydrolytically degradable. It has also been examined as a material for drug delivery devices.^127,131-134 Polycarbonates have been suggested as suitable materials to make scaffolds for bone replacement and have been modified with tyrosine-derived amino acids to render them biodegradable.¹²⁷ Polydioxanone has been also tested for biomedical applications.¹³⁵ PMMA is widely used in orthopedics as a bone cement for implant fixation as well as to repair certain fractures and bone defects, for example, osteoporotic vertebral bodies.^136,137 However, PMMA sets via a polymerization of toxic monomers, which also produces a significant amount of heat that damages tissues. Moreover, it is neither degradable nor bioactive, does not bond chemically to bones and might generate particulate debris, leading to an inflammatory foreign body response.^131,138 A number of other nondegradable polymers applied in orthopedic surgery include PE in its different modifications, such as low density PE, HDPE and UHMWPE (used as the articular surface of total hip replacement implants^139,140), polyethylene terepthalate, PP and PTFE, which are applied to repair knee ligaments.¹⁴¹ Polyactive^TM, a block copolymer of PEG and PBT, has also been considered for biomedical application.^142-147 Cellulose^148,149 and its esters^150,151 are also popular. Finally, and importantly, polyethylene oxide, PHB and blends thereof have also been tested for biomedical applications.⁵⁰

Nonetheless, the most popular synthetic polymers used in medicine are the linear aliphatic poly(α-hydroxyesters), such as PLA, PGA and their copolymers, PLGA (Table 4). These materials have been extensively studied; they appear to be the only synthetic and biodegradable polymers with an extensive FDA approval history.^{50,132,152-156} They are biocompatible, mostly non-inflammatory and can degrade in vivo through hydrolysis and, possibly, enzymatic action into products that are removed from the body by regular metabolic pathways.^{49,127,132,156-161} They might also be used for drug delivery purposes.¹⁶² Poly(α-hydroxyesters) have been investigated as scaffolds for replacement and regeneration of a variety of tissues, cell carriers, controlled delivery devices for drugs or proteins (e.g., growth factors), membranes or films, screws, pins and plates for orthopedic applications.^{127,132,153,154,156,163-165} Additionally, the degradation rate of PLGA can be adjusted by varying the amounts of the two component monomers (Table 4), which in orthopedic applications can be exploited to create materials that degrade in concert with bone ingrowth.^160,166 Furthermore, PLGA is known to support osteoblast migration and proliferation,^{59,132,157,167} which is a necessity for bone tissue regeneration. Unfortunately, such polymers on their own, though they reduce the effect of stress shielding, are too weak to be used in load-bearing situations and are only recommended in certain clinical indications, such as ankle and elbow fractures.^156,161 In addition, they exhibit bulk degradation, leading to both a loss in mechanical properties and lowering of the local solution pH, which further accelerates degradation in an autocatalytic manner. As the body is unable to cope with the vast amounts of implant degradation products, this might lead to an inflammatory foreign body response.^132,156,163 Finally, poly(α-hydroxyesters) do not possess the bioactive and osteoconductive properties of calcium orthophosphates.^153,168

Table 4. Major properties of several FDA approved biodegradable polymers.¹⁵² .

polymer	thermal properties*, °C	tensile modulus, GРa	degradation time, months
polyglycolic acid (PGA)	tg = 35 – 40 tm = 225 – 230	7.06	6 – 12 (strength loss within 3 weeks)
L-polylactic acid (LPLA)	tg = 60 – 65 tm = 173 – 178	2.7	> 24
D,L-polylactic acid (DLPLA)	tg = 55 – 60 amorphous	1.9	12 – 16
85/15 D,L-polylactic-co-glycolic acid (85/15 DLPLGA)	tg = 50 – 55 amorphous	2.0	5 – 6
75/25 D,L-polylactic-co-glycolic acid (75/25 DLPLGA)	tg = 50 – 55 amorphous	2.0	4 – 5
65/35 D,L-polylactic-co-glycolic acid (65/35 DLPLGA)	tg = 45 – 50 amorphous	2.0	3 – 4
50/50 D,L-polylactic-co-glycolic acid (50/50 DLPLGA)	tg = 45 – 50 amorphous	2.0	1 – 2
poly(ε-caprolactone) (PCL)	tg = (– 60) – (– 65) tm = 58 – 63	0.4	> 24

^*t_g – glass transition temperature; t_m – melting point.

Several classifications of the biomedically relevant polymers are possible. For example, some authors distinguish between synthetic polymers, like PLA, PGA or their copolymers, and PCL and polymers of biological origin like polysaccharides (starch, alginate, chitin/chitosan,^169-171 gelatin, cellulose, hyaluronic acid derivatives), proteins (soy, collagen, fibrin,¹¹ silk) and a variety of biofibers, such as lignocellulosic natural fibers.^10,172,173 Natural polymers often possess highly organized structures and may contain an extracellular substance, called ligand, which is necessary to bind with cell receptors. However, they always contain various impurities that should be gotten rid of prior to use. As synthetic polymers can be produced under controlled conditions, in general, they exhibit predictable and reproducible mechanical and physical properties, such as tensile strength, elastic modulus and degradation rate. Control of impurities is a further advantage of synthetic polymers. Other authors differentiate between resorbable or biodegradable [e.g., poly(α-hydroxyesters), polysaccharides and proteins] and non-resorbable (e.g., PE, PP, PMMA and cellulose) polymers.^60,173 Furthermore, polymeric materials can be broadly classified as thermoplastics and thermosets. HDPE and PEEK are examples of thermoplastics, while polydimethylsiloxane and PMMA are the examples of thermosets.¹²² The list of synthetic biodegradable polymers used for biomedical application as scaffold materials is available as Table 1 in reference ,173 while further details on polymers suitable for biomedical applications are available in the literature (refs.122,165,174–183), where interested readers are referred. Good reviews on the synthesis of different biodegradable polymers¹⁸⁴ as well as on the experimental trends in polymer composites¹⁸⁵ are available elsewhere.

Inorganic materials and compounds

Metals

Titanium (Ti) is one of the best biocompatible metals and is used most widely as an implant.^16,186,187 Besides Ti, there are other metallic implants made of pure Zr, Hf, V, Nb, Ta, Re,¹⁸⁶ Ni, Fe, Cu,^188-190 Ag, stainless steels and various alloys¹⁹⁰ suitable for biomedical application. Recent studies revealed an even greater biomedical potential for porous metals.^191-194 Metallic implants provide the necessary strength and toughness required in load-bearing parts of the body, and, due to these advantages, metals will continue to play an important role as orthopedic biomaterials in the future, even though there are concerns with regard to the release of certain ions from and corrosion products of metallic implants. Of course, neither metals nor alloys are biomimetic (the term biomimetic can be defined as a processing technique that either mimics or inspires the biological mechanism, in part or whole¹⁹⁵) in terms of chemical composition, because there are no elemental metals in the human body. In addition, even biocompatible metals are bioinert; although they are not rejected by the human body, metallic implants cannot actively interact with the surrounding tissues. Nevertheless, in some cases (especially when they are coated by calcium orthophosphates; however, that is another story), the metallic implants show a reasonable biocompatibility.¹⁹⁶ Only permanent implants are made of metals and alloys, in which degradation or corrosion is not desirable. However, in recent years, a number of magnesium implants have been proposed which are aimed to degrade in the body in order to make room for ingrowing bones.^193,197,198

Glasses and glass-ceramics

Special types of glasses and glass-ceramics are also suitable materials for biomedical applications,^199-201 and a special Na₂O-CaO-SiO₂-P₂O₅ glass, named Bioglass^®,^{13,28,30,31,202,203} is the most popular among them. They are produced via standard glass production techniques and require pure raw materials. Bioglass^® is a biocompatible and osteoconductive biomaterial. It bonds to bone without an intervening fibrous connective tissue interface and, due to these properties, it has been widely used for filling bone defects.²⁰⁴ The primary shortcoming of Bioglass^® is mechanical weakness and low fracture toughness due to an amorphous two-dimensional glass network. The bending strength of most Bioglass^® compositions is in the range of 40–60 MPa, which is not suitable for major load-bearing applications. Making porosity in Bioglass^®-based scaffolds is beneficial, even for better resorption and bioactivity.²⁰⁵

By heat treatment, a suitable glass can be converted into glass-crystal composites containing crystalline phase(s) of controlled sizes and contents. The resultant glass-ceramics can have superior mechanical properties to the parent glass as well as to sintered crystalline ceramics. The bioactive A-W glass-ceramics are made from the parent glass in the pseudoternary system 3CaO·P₂O₅-CaO·SiO₂-MgO·CaO·2SiO₂, which is produced by a conventional melt-quenching method. The bioactivity of A-W glass-ceramics is much higher than that of sintered HA. They possess excellent mechanical properties and have, therefore, been used clinically for iliac and vertebrae prostheses and as intervertebral spacers.^16,206-208

Ceramics

Metal oxide ceramics, such as alumina (Al₂O₃, high purity, polycrystalline, fine grained), zirconia (ZrO₂) and some other oxides (e.g., TiO₂, SiO₂) have been widely studied due to their bioinertness, excellent tribological properties, high wear resistance, fracture toughness and strength as well as relatively low friction.^16,209 Unfortunately, due to transformation from the tetragonal to the monoclinic phase, a volume change occurs when pure zirconia is cooled down, which causes cracking of the zirconia ceramics. Therefore, additives such as calcia (CaO), magnesia (MgO) and yttria (Y₂O₃) must be mixed with zirconia to stabilize the material in either the tetragonal or the cubic phase. Such material is called PSZ.^210-212 However, the brittle nature of ceramics has limited their scope of clinical applications, and hence, more research needs to be conducted to improve their properties.

Carbon

Due to its bioinertness, excellent tribological properties, fracture toughness and strength as well as low friction, elemental carbon has been used as a biomaterial at least since 1972.²¹³ Applications include orthopedic prostheses, vitreous carbon roots for replacement teeth, structural skeletal extensions, bone bridges and hip prostheses. Biomedical properties of amorphous carbon were studied as well.²¹⁴ However, current trends primarily represent investigations on biomedical applications of carbon nanotubes.^215,216

Carbon nanotubes, with their small dimensions, high aspect (length to diameter) ratio as well as exceptional mechanical properties, including extreme flexibility and strength, significant resistance to bending, high resilience and the ability to reverse any buckling of the tube, have excellent potential for accomplishing the necessary mechanical properties.²¹⁷ Recent studies have even suggested that they may possess some bioactivity.^218-221 However, non-functionalized carbon nanotubes tend to agglomerate and form bundles. Besides, they are soluble in neither water nor organic solvents. Luckily, chemical functionalization^82,222 allows carbon nanotubes to be dispersed more easily, which can improve interfacial bonding with other components of the composites. Furthermore, functionalization of carbon nanotubes with carboxylic groups was found to confer a capacity to induce calcification similar to woven bones.²²³ Interestingly, carbon nanotubes might be functionalized by in situ deposition of CDHA on their surface.²²⁴

Biocomposites and Hybrid Biomaterials Based on Calcium Orthophosphates

Generally, the available biocomposites and hybrid biomaterials based on calcium orthophosphates might be divided into several (partly overlapping) broad areas:

• biocomposites with polymers,

• self-setting formulations and concretes,

• formulations based on nanodimensional calcium orthophosphates and nanodimensional biocomposites,

• biocomposites with collagen,

• formulations with other bioorganic compounds and/or biological macromolecules,

• injectable bone substitutes (IBS),

• biocomposites with glasses, inorganic compounds, carbon and metals,

• functionally graded formulations and

• biosensors

The details on each subject are discussed below.

Biocomposites with polymers

Typically, the polymeric components of biocomposites and hybrid biomaterials comprise polymers that have shown both a good biocompatibility and are routinely used in surgical applications. In general, since polymers have a low modulus (2–7 GPa, as the maximum) as compared with that of bone (3–30 GPa), calcium orthophosphate bioceramics need to be loaded at a high weight % ratio. Besides, general knowledge on composite mechanics suggests that any high aspect ratio particles, such as whiskers or fibers, significantly improve the modulus at a lower loading.¹⁷⁹ Thus, some attempts have already been made to prepare biocomposites containing whisker-like^225-229 or needle-like^230-232 calcium orthophosphates as well as calcium orthophosphate fibers.^49,233

The history of implantable polymer-calcium orthophosphate biocomposites and hybrid biomaterials started in 1981²³⁴ with the pioneering study by Prof. William Bonfield and colleagues performed on HA/PE formulations.^236,237 That initial study introduced a bone analog concept, in which proposed biocomposites comprised a polymer ductile matrix of PE and a ceramic stiff phase of HA that was substantially extended and developed in further investigations by that research group.^102,238-254 More recent studies have included investigations on the influence of surface topography of HA/PE composites on cell proliferation and attachment.^255-261 The material is composed of a particular combination of HA particles at a volume loading of ~40% uniformly dispensed in a HDPE matrix. Alternatively, PP might be used instead of PE.^262-264 The idea was to mimic bones by using a polymeric matrix that can develop a considerable anisotropic character through adequate orientation techniques, reinforced with a bone-like bioceramic material that assures both a mechanical reinforcement and a bioactive character of the composite. Following FDA approval in 1994, in 1995 this material became commercially available under the trade name HAPEX^TM (Smith and Nephew, Richards), and, to date, it has been implanted in over 300,000 patients with successful results. It remains the only clinically successful bioactive composite and appeared to be a major step in the implant field.^31,265 The major production stages of HAPEX^TM include blending, compounding and centrifugal milling. A bulk material or device is then created from this powder by compression and injection molding.⁶³ Alternatively, HA/HDPE biocomposites might be prepared by a hot rolling technique that facilitates uniform dispersion and blending of the reinforcements in the matrix.²⁶⁶

A mechanical interlock between the two phases of HAPEX^TM is formed by the shrinkage of HDPE onto the HA particles during cooling.^102,267 Both HA particle size and their distribution in the HDPE matrix are recognized as important parameters affecting the mechanical behavior of HAPEX^TM.²⁴⁷ Smaller HA particles, for example, were found to lead to stiffer composites due to the increase in interfaces between the polymer and the ceramics. In addition, rigidity of HAPEX^TM was found to be proportional to HA volume fraction.²³⁹ Coupling agents, e.g., 3-trimethoxysiyl propylmethacrylate for HA and acrylic acid for HDPE, might be used to improve bonding (by both chemical adhesion and mechanical coupling) between HA and HDPE.^268,269 Obviously, other calcium orthophosphates might be used instead of HA in biocomposites with PE.²⁷⁰ Indeed, attempts were made to improve the mechanical properties of HAPEX^TM by incorporating other ceramic phases into the polymer matrix, such as PSZ²⁷¹ and alumina.²⁷² A partial replacement of HA filler particles by PSZ particles was found to lead to an increase in the strength and fracture toughness of HA/HDPE biocomposites. The compressive stress, set up by the volume expansion associated with the tetragonal-to-monoclinic phase transformation of PSZ, inhibits or retards the crack propagation within the composite. This results in an enhanced fracture toughness of the HA/ZrO₂/HDPE biocomposite.²⁷¹

Various studies revealed that HAPEX^TM attached directly to bones by chemical bonding (a bioactive fixation) rather than by forming fibrous encapsulation (a morphological fixation). Initial clinical applications of HAPEX^TM came in orbital reconstruction,²⁷³ but since 1995, the main uses of this composite have been in the shafts of middle ear implants for the treatment of conductive hearing loss.^274,275 In both applications, HAPEX^TM offers the advantage of in situ shaping, so a surgeon can make final alterations to optimize the fit of the prosthesis to the bone of a patient, and subsequent activity requires only limited mechanical loading with virtually no risk of failure from insufficient tensile strength.^102,202 As compared with cortical bones, HA/ PE composites have a superior fracture toughness for HA concentrations below ~40% and similar fracture toughness in the 45–50% range. Their Young’s modulus is in the range of 1–8 GPa, which is quite close to that of bone. The examination of the fracture surfaces revealed that only a mechanical bond occurs between HA and PE. Unfortunately, the HA/PE composites are not biodegradable, the available surface area of HA is low, and the presence of bioinert PE decreases the ability to bond to bones. Furthermore, HAPEX^TM has been designed with a maximized density to increase its strength, but the resulting lack of porosity limits the ingrowth of osteoblasts when the implant is placed into the body.^29,203 Further details on HAPEX^TM are available elsewhere.¹⁰² In addition to HAPEX^TM, other types of HA/PE biocomposites are also known.^276-282

Both linear and branched PE were used as a matrix, and the biocomposites with the former were found to give a higher modulus.²⁷⁷ The reinforcing mechanisms in calcium orthophosphate/polymer biocomposites have yet to be convincingly disclosed. Generally, if a poor filler choice is made, the polymeric matrix might be affected by the filler through reduction of molecular weight during composite processing, formation of an immobilized shell of polymer around the particles (transcrystallization, surface-induced crystallization or epitaxial growth) and changes in conformation of the polymer due to particle surfaces and inter-particle spacing.¹⁰² On the other hand, the reinforcing effect of calcium orthophosphate particles might depend on the molding technique employed: a higher orientation of the polymeric matrix was found to result in a higher mechanical performance of the composite.^282,283

Many other blends of calcium orthophosphates with various polymers are possible, including rather unusual formulations with dendrimers.²⁸⁴ Even light-curable polymer/calcium orthophosphate formulations are known.²⁸⁵ The list of the appropriate calcium orthophosphates is shown in Table 3 (except MCPM and MCPA, as both are too acidic and, therefore, are not biocompatible;²⁷ however, to overcome this drawback, they might be mixed with basic compounds, such as HA, TTCP, CaCO₃, CaO, etc.). Many biomedically suitable polymers have been listed above. The combination of calcium orthophosphates and polymers into biocomposites has a 2-fold purpose. The desirable mechanical properties of polymers compensate for a poor mechanical behavior of calcium orthophosphate bioceramics, while, in turn, the desirable bioactive properties of calcium orthophosphates improve those of polymers, expanding the possible uses of each material within the body.^{158-160,286-290} Namely, polymers have been added to calcium orthophosphates in order to improve their mechanical strength,^158,286 and calcium orthophosphate fillers have been blended with polymers to improve their compressive strength and modulus in addition to increasing their osteoconductive properties.^{52,160,168,291-295} Furthermore, biocompatibility of such biocomposites is enhanced, because calcium orthophosphate fillers induce an increased initial flash spread of serum proteins compared with the more hydrophobic polymer surfaces.²⁹⁶ What’s more, experimental results of these biocomposites indicate favorable cell-material interactions with increased cell activities as compared with each polymer alone.²⁸⁸ As a rule, with increasing of calcium orthophosphate content, both Young’s modulus and bioactivity of the biocomposites increase, while the ductility decreases.^29,291 Furthermore, such formulations can provide a sustained release of calcium and orthophosphate ions into the milieus, which is important for mineralized tissue regeneration.²⁸⁷ Indeed, a combination of two different materials draws on the advantages of each one to create a superior biocomposite with respect to the materials on their own.

It is logical to assume that the proper biocomposite of a calcium orthophosphate (for instance, CDHA) with a bioorganic polymer (for instance, collagen) would yield physical, chemical and mechanical properties similar to those of human bones. Different methods for bringing these two components together into biocomposites have already been realized, including mechanical blending, compounding, ball milling, dispersion of ceramic fillers into a polymer-solvent solution, a melt extrusion of a ceramic/polymer powder mixture, coprecipitation and electrochemical co-deposition.^{36,63,297-299} Three methods for preparing a homogeneous blend of HA with PLLA were compared.²⁹⁷ First, a dry process, consisting of mixing ceramic powder and polymer pellets before a compression molding step, was used. The second technique was based on the dispersion of ceramic fillers into a polymer-solvent solution. The third method was a melt extrusion of a ceramic/polymer powder mixture. Mixing dry powders led to a ceramic particle network around the polymer pellets, whereas the solvent and melt methods produced a homogeneous dispersion of HA in the matrix. The main drawback of the solvent casting method is the risk of potentially toxic organic solvent residues. The melt extrusion method was shown to be a good way to prepare homogeneous ceramic/polymer blends.²⁹⁷

There is also in situ formation, which involves either synthesizing the reinforcement inside a preformed matrix material or synthesizing the matrix material around the reinforcement.^63,300,301 This is one of the most attractive routes, since it avoids extensive particle agglomeration. Several papers have reported that the in situ formation technique has produced various composites of apatites with carbon nanotubes.^302-308 Other appoaches include using amino acid-capped nano-sized gold particles as scaffolds to grow CDHA³⁰⁹ and in preparation of nano-sized HA/polyamide biocomposites.^310,311 In certain cases, a mechanochemical route,³¹² emulsions,^313-316 freeze-drying³¹⁷ and freeze-thawing techniques,³¹⁸ flame-sprayed technique³¹⁹ or gel-templated mineralization³²⁰ might be applied to produce calcium othophosphates-based biocomposites. Various fabrication procedures are well described elsewhere in references 36, 63 and 297 where the interested readers are referred.

The interfacial bonding between a calcium orthophosphate and a polymer is an important issue for any biocomposite. Four types of mutual arrangements of nanodimensional particles to polymer chains have been classified by Kickelbick (Fig. 1): (1) inorganic particles embedded in inorganic polymer, (2) incorporation of particles by bonding to the polymer backbone, (3) an interpenetrating network with chemical bonds and (4) an inorganic-organic hybrid polymer.³²¹ If adhesion among the phases is poor, the mechanical properties of a biocomposite suffer. To solve the problem, various approaches have been already introduced. For example, a diisocyanate coupling agent was used to bind PEG/PBT (Polyactive^TM) block copolymers to HA filler particles. Using surface-modified HA particles as a filler in a PEG/PBT matrix significantly improved the elastic modulus and strength of the polymer as compared with the polymers filled with ungrafted HA.^293,322 Another group used processing conditions to achieve a better adhesion of the filler to the matrix. Ignjatovic et al. prepared PLLA/HA composites by pressing blends of varying PLLA and HA content at different temperatures and pressures.^158,159,323 They found that maximum compressive strength was achieved at ~15 wt% of PLLA. By using blends with 20 wt% of PLLA, the authors also established that increasing the pressing temperature and pressure improved the mechanical properties. The former was explained by a decrease in viscosity of the PLLA associated with a temperature increase, hence leading to improved wettability of HA particles. The latter was explained by increased compaction and penetration of pores at higher pressure in conjunction with a greater fluidity of the polymer at higher temperatures. The combination of high pressures and temperatures was found to decrease porosity and guarantee a close apposition of a polymer to the particles, thereby improving the compressive strength²⁸⁶ and fracture energy³²⁴ of the biocomposites. The PLLA/HA biocomposites’ scaffolds were found to improve cell survival over plain PLLA scaffolds.³²⁵

Figure 1.

Four types of mutual arrangements of nano-sized particles to a polymer chain: (1) inorganic particles embedded in an inorganic polymer, (2) incorporation of particles by bonding to the polymer backbone, (3) interpenetrating network with chemical bonds, (4) inorganic-organic hybrid polymer. Reprinted from reference 321 with permission.

It is also possible to introduce porosity into calcium orthophosphate-based biocomposites, which is advantageous for most applications as bone substitution material. The porosity facilitates migration of osteoblasts from surrounding bones to the implant site.^160,326,327 Various material processing strategies to prepare composite scaffolds with interconnected porosity comprise thermally induced phase separation, solvent casting and particle leaching, solid freeform fabrication techniques, microsphere sintering and coating.^173,328-330 A supercritical gas foaming technique might be used as well.^297,331,332

Apatite-based formulations

A biological apatite is known to be the major inorganic phase of mammalian calcified tissues.^25,26 Consequently, CDHA, HA, carbonateapatite (both with and without dopants) and, occasionally, FA have been applied to prepare biocomposites with other compounds, usually with the aim of improving the bioactivity. For example, PS composed with HA can be used as a starting material for long-term implants.^333-335 Retrieved in vivo, HA/PS biocomposite-coated samples from rabbit distal femurs demonstrated direct bone apposition to the coatings as compared with the fibrous encapsulation that occurred when uncoated samples were used.³³³ The resorption time of such biocomposites is a very important factor, which depends on polymer’s microstructure and the presence of modifying phases.³³⁴

Various apatite-containing biocomposites with PVA,^318,336-344 PVAP³⁴⁵ and several other polymeric components^346-358 have already been developed. Namely, PVA/CDHA biocomposite blocks were prepared by precipitation of CDHA in aqueous solutions of PVA.³¹⁸ An artificial cornea consisting of a porous nano-sized HA/PVA hydrogel skirt and a transparent center of PVA hydrogel has been prepared as well. The results displayed good biocompatibility and interlocking between artificial cornea and host tissues.^340,341 PVAP has been chosen as a polymer matrix, because its phosphate groups can act as a coupling/anchoring agent with a higher affinity toward the HA surface.³⁴⁵ Greish and Brown developed HA/Ca poly(vinyl phosphonate) biocomposites.^349-351 A template-driven nucleation and mineral growth process for the high-affinity integration of CDHA with PHEMA hydrogel scaffold has been developed as well.³⁵⁸

PEEK^{225,227,359-365,367} and HIPS³⁶⁶ were applied to create biocomposites with HA because of their potential for clinical use in load-bearing applications. The study on reinforcing PEEK with thermally sprayed HA particles revealed that the mechanical properties increased monotonically with the reinforcement concentration, with a maximum value in the study of ~40% volume fraction of HA particles.^361-363 The reported ranges of stiffness within 2.8–16.0 GPa and strength within 45.5–69 MPa exceeded the lower values for human bone (7–30 GPa and 50–150 MPa, respectively).³⁶² Modeling of the mechanical behavior of HA/ PEEK biocomposites is available elsewhere.³⁶⁴

Biodegradable poly(α-hydroxyesters) are well established in clinical medicine. Currently, they provide a good choice when a suitable polymeric filler material is sought. For example, HA/ PLGA composites have been developed that appear to possess a cellular compatibility suitable for bone tissue regeneration.^368-376 Zhang and Ma seeded highly porous PLLA foams with HA particles in order to improve the osteoconductivity of polymer scaffolds for bone tissue engineering.^52,292 They pointed out that hydration of the foams prior to incubation in simulated body fluid increased the amount of carbonated CDHA material due to an increase of COOH and OH groups on the polymer surface, which apparently acted as nucleation sites for apatite. The following values of Young’s modulus, compressive, bending and tensile strengths for PLLA/HA composites have been achieved: 5–12 GPa, 78–137 MPa, 44–280 MPa and 10–30 MPa, respectively.³⁷⁷ However, these data do not appear to be in a good agreement with HA/PLLA biocomposite unit cell model predictions.³⁷⁸

On their own, PGA and PLA are known to degrade to acidic products (glycolic and lactic acids, respectively) that both catalyze polymer degradation and cause inflammatory reactions of the surrounding tissues.³⁷⁹ However, in biocomposites of poly(α-hydroxyesters) with calcium orthophosphates, the presence of slightly basic compounds (HA, TTCP) neutralizes the acid molecules to some extent and provides a weak pH-buffering effect at the polymer surface, therefore more or less compensating for their drawbacks.^168,380-382 However, additives of even more basic chemicals (e.g., CaO, CaCO₃) might be necessary.^{173,381,383,384} Extensive cell culture experiments on pH-stabilized composites of PGA and carbonateapatite were reported, which afterwards were supported by extensive in vitro pH studies.³⁸⁵ A consequent development of this approach has led to the designing of functionally graded composite skull implants consisting of polylactides, carbonateapatite and CaCO₃.^386,387 Besides the pH-buffering effect, inclusion of calcium orthophosphates was found to modify both surface and bulk properties of the biodegradable poly(α-hydroxyesters) by increasing the hydrophilicity and water absorption of the polymer matrix, thus altering the scaffold degradation kinetics. For example, polymer biocomposites filled with HA particles were found to hydrolyze homogeneously due to water penetrating into interfacial regions.³⁸⁸

Biocomposites of poly(α-hydroxyesters) with calcium orthophosphates are prepared mainly by incorporating the inorganic phase into a polymeric solution followed by drying under vacuum. The resulting solid biocomposites might be shaped using different processing techniques. One can also prepare these biocomposites by mixing HA particles with L-lactide prior to the polymerization³⁸⁰ or by a combination of a slip-casting technique and hot pressing.³⁸⁹ Addition of a surfactant (surface active agent) might be useful to keep the suspension homogenous.³⁹⁰ Furthermore, HA/PLA^314,315 and HA/PLGA³¹⁶ microspheres might be prepared by a microemulsion technique. More complex carbonated FA/PLA³⁹¹ and PLGA/carbon nanotubes/HA³⁹² porous biocomposite scaffolds are also known. An interesting list of references assigned to the different ways of preparing HA/poly(α-hydroxyesters) biodegradable composites might be found in publications by Durucan and Brown.^53,393,394 The authors prepared CDHA/PLA and CDHA/PLGA biocomposites using a solvent casting technique with a subsequent hydrolysis of α-TCP to CDHA in aqueous solutions. The presence of both polymers was found to inhibit α-TCP hydrolysis compared with that of single-phase α-TCP alone; what’s more, the inhibiting effect of PLA exceeded that of PLGA.^53,393,394 The physical interactions between calcium orthophosphates and poly(α-hydroxyesters) might be easily seen in Figure 2.⁵³ Another set of good pictures might be found in reference 87. Nevertheless, it should not be forgotten that, typically, non-melt-based routes lead to the development of composites with lower mechanical performance and often times require the use of toxic solvents and intensive hand labor.¹⁷⁸

Figure 2.

SEM micrographs of (A) α-TCP compact; (B) α-TCP/PLGA biocomposite (bars = 5 µm). Reprinted from reference 53 with permission.

The mechanical properties of poly(α-hydroxyesters) could be substantially improved by the addition of calcium orthophosphates.^395,396 Shikinami and Okuno developed CDHA/PLLA composites with very high mechanical properties;¹⁶⁸ mini-screws and mini-plates made of these composites have been manufactured and tested.³⁸⁸ They have shown easy handling and shaping according to the implant site geometry, total resorbability, good ability to bond directly to the bone tissue without interposed fibrous tissue, osteoconductivity, biocompatibility and high stiffness that can be retained for the period necessary to achieve bone union.³⁸⁸ The initial bending strength of ~280 MPa exceeded that of cortical bone (120–210 MPa), while the modulus was as high as 12 GPa.¹⁶⁸ The strength could be maintained above 200 MPa up to 25 weeks in phosphate-buffered saline solution. Such biocomposites were obtained from precipitation of a PLLA/dichloromethane solution, where small granules of uniformly distributed CDHA microparticles (average size of 3 µm) could be prepared.¹⁶⁷ Porous scaffolds of PDLLA and HA have been manufactured as well.^332,397,398 Upon implantation into rabbit femora, a newly formed bone was observed, and biodegradation was significantly enhanced compared with single-phase HA bioceramics. This might be due to a local release of lactic acid, which, in turn, dissolves HA. In other studies, PLA and PGA fibers were combined with porous HA scaffolds. Such reinforcement did not hinder bone ingrowth into the implants, which supported further development of such biocomposites as bone graft substitutes.^{50,51,377,399,400}

Blends (named SEVA-C) of EVOH with starch filled with 10–30 wt% HA have been fabricated to yield biocomposites with moduli up to ~7 GPa and a 30% HA loading.^401-406 The incorporation of bioactive fillers, such as HA into SEVA-C, aimed to insure the bioactive behavior of the composite and to provide the necessary stiffness within the typical range of human cortical bone properties. These biocomposites exhibited a strong in vitro bioactivity, which was supported by the polymer’s water-uptake capability.⁴⁰⁷ However, the reinforcement of SEVA-C by HA particles was found to affect the rheological behavior of the blend. A degradation model of these biocomposites has been developed.⁴⁰⁸

Higher homologs poly(3-hydroxybutyrate), 3-PHB and poly(3-hydroxyvalerate), 3-PHV, show almost no biodegradation. Nevertheless, biocomposites of these polymers with calcium orthophosphates show a good biocompatibility both in vitro and in vivo.^102,409-415 Both bioactivity and mechanical properties of these biocomposites can be tailored by varying the volume percentage of calcium orthophosphates. Similarly, biocomposites of PHBHV with both HA and amorphous carbonated apatite (almost ACP) appeared to have promising potential for repair and replacement of damaged bones.^416-419

Along these lines, PCL is used as a slowly biodegradable but good biocompatible polymer. PCL/HA and PCL/CDHA biocomposites have already been discussed as suitable materials for substitution, regeneration and repair of bone tissues.^328,420-433 For example, biocomposites were obtained by infiltration of Σ-caprolactone monomer into porous apatite blocks and by in situ polymerization.⁴²³ The composites were found to be biodegradable and might be applied as cancellous or trabecular bone replacement material or for cartilage regeneration. Both the mechanical performance and biocompatibility in osteoblast cell culture of PCL were shown to be strongly increased when HA was added.⁴³⁴ Several preparation techniques of PCL/HA biocomposites are known. For example, to make biocomposite fibers of PCL with nanodimensional HA, the desired amount of nanodimensional HA powder was dispersed in a solvent using magnetic stirrer, followed by ultrasonication for 30 min. Then, PCL was dissolved in this suspension, followed by solvent evaporation.⁴³⁵ The opposite preparation order has also been used: PCL was initially dissolved in chloroform at room temperature (7–10% weight/volume), then HA (~10 µm particle size) was suspended in the solution, sonicated for 1 min, followed by solvent evaporation¹⁶⁰ or salt-leaching.⁴³⁶ The mechanical properties obtained by this technique were about one-third that of trabecular bone. In a comparative study, PCL and biological apatite were mixed in a 19:1 ratio in an extruder.⁴³⁷ At the end of the preparation, the mixture was cooled in an atmosphere of nitrogen. The authors observed that the presence of biological apatite improved the modulus, while concurrently increasing the hydrophilicity of the polymeric substrate. In addition, an increase in apatite concentration was found to increase both the modulus and yield stress of the composite, which indicated good interfacial interactions between the biological apatite and PCL. It was also observed that the presence of biological apatite stimulated osteoblasts’ attachment to the biomaterial and cell proliferation.⁴³⁷ In another study, a PCL/HA biocomposite was prepared by blending in melt form at 120°C until the torque reached equilibrium in the rheometer that was attached to the blender.⁴³⁸ Then the sample was compression molded and cut into specimens of appropriate size for testing. It was observed that the composite containing 20 wt% HA had the highest strength.⁴³⁸ However, a direct grafting of PCL onto the surface of HA particles seems to be the most interesting preparation technique.⁴²⁰ In another study, HA porous scaffolds were coated by a PCL/HA composite coating.⁵⁴ In this system, PCL as a coating component was able to improve the brittleness and low strength of the HA scaffolds, while the particles in the coating improved the osteoconductivity and bioactivity of the coating layer. More complex formulations, such as PDLLA/PCL/HA,⁴³⁹ PLLA/PCL/HA⁴⁴⁰ and supramolecular PCL/functionalized HA⁴⁴¹ biocomposites, have been prepared as well. Further details on both the PCL/HA biocomposites and the processing methodologies thereof might be found in reference 328.

A spread of human osteoblasts attached to PLA and PCL films reinforced with CDHA and sintered HA was shown to have higher stength than the polymers alone.¹⁸⁴ Moreover, biochemical assays relating cell activity to DNA content allowed for the conclusion that cell activity was more intense for the composite films.¹⁸⁴ Kim et al. coated porous HA blocks with PCL from dichloromethane solution and performed drug release studies. The antibiotic tetracycline hydrochloride was added into this layer, yielding a bioactive implant with drug release for longer than a week.⁵⁴

Yoon et al. investigated the highest mechanical and chemical stability of FA by preparing FA/collagen biocomposites and studying their effect on osteoblast-like cell culture.⁴⁴² The researchers found an increased cellular activity in FA composites compared with HA composites. This finding was confirmed in another study by means of variations in the fluoride content for FA-HA/PCL composites.⁴⁴³ An interesting phenomenon of fractal growth of FA/gelatin composite crystals (Fig. 3) was achieved by diffusion of calcium^- and orthophosphate⁺ fluoride solutions from opposite sides into a tube filled with a gelatin gel.^444-453 The reasons for this phenomenon are not quite clear yet; besides, up to now, nothing has yet been reported on a possible biomedical application for such very unusual structural composites.

Figure 3.

A biomimetically grown aggregate of FA that was crystallized in a gelatin matrix. Its shape can be explained and simulated by a fractal growth mechanism. Scale bar: 10 µm. Reprinted from reference 444 with permission.

TCP-based formulations

Both α-TCP and β-TCP have a higher solubility than HA (Table 3), and they are resorbed more quickly in vivo.⁴⁵⁴ Therefore, these calcium orthophosphates were widely used instead of apatites to prepare completely biodegradable biocomposites.^456-479 For example, a biodegradable and osteoconductive biocomposite made of β-TCP particles and gelatin was proposed.⁴⁶⁶ This material was tested in vivo with good results. It was found to be biocompatible, osteoconductive and biodegradable, with no need for a second surgical operation to remove the device after healing occurred. Both herbal extracts⁴⁶⁷ and K₂HPO₄⁴⁶⁸ might be added to this formulation. Another research group prepared biocomposites of cross-linked gelatin with β-TCP, and both a good biocompatibility and bone formation upon subcutaneous implantation in rats were found.⁴⁶⁹ Yang et al.⁴⁷⁴ extended this to porous (porosity ~75%) β-TCP/gelatin biocomposites that also contained BMP-4. Porous β-TCP/alginate-gelatin hybrid scaffolds that were cell-compatible and possessing some osteoinductive properties were aso prepared and successfully tested in vitro.⁴⁷¹ Biocomposites of β-TCP with PLLA^462-464 and PLGC⁴⁶⁵ were prepared as well. Although β-TCP was able to counter the acidic degradation of the polyester to some extent, it did not prevent a pH drop down to ~6. Nevertheless, implantation of this biocomposite in beagles’ mandibular bones was successful.⁴⁶⁵ α-TCP/gelatin formulations are known as well.⁴⁷⁷

Based on a self-reinforcement concept, biocomposites of TCP with polylactides were prepared and studied using conventional mechanical testing.⁴⁸⁰ Resorbable scaffolds were fabricated from such biocomposites.⁴⁸¹ Chitosan was used as the matrix for the incorporation of β-TCP by a solid/liquid phase separation of the polymer solution and subsequent sublimation of the solvent. Due to complexation of the functional groups of chitosan with calcium ions of β-TCP, these biocomposites had a high compressive modulus and strength.⁴⁸² PCL/β-TCP biocomposites were developed in other studies,^483-486 and their in vitro degradation behavior was systematically monitored by immersion in simulated body fluid at 37°C.⁴⁸⁵ To extend this topic further, PCL/β-TCP biocomposites might be loaded by drugs.⁴⁸⁶

Cell culture tests on β-TCP/PLLA biocomposites were reported; the biocomposites showed no cytotoxicity and evidenced good cell attachment to its surface.⁴⁵⁶ An in vitro study with primary rat calvarial osteoblasts showed an increased cellular activity in the BMP-loaded samples.⁴⁷⁴ Other researchers investigated BMP-2-loaded porous β-TCP/gelatin biocomposites (porosity ~95%, average pore size 180–200 µm)⁴⁸⁷ and confirmed the results of the previous study. Biocomposites of β-TCP and glutaraldehyde cross-linked gelatin were manufactured and tested in vitro to measure the material cytotoxicity.⁴⁷⁰ The experimental results revealed that the amount of glutaraldehyde cross-linking agent should be less than 8% to decrease toxicity on the osteoblasts and to avoid inhibition of cellular growth caused by the release of residual or un-cross-linked glutaraldehyde. A long-term implantation study of PDLLA/ α -TCP composites in a loaded sheep implant model showed good results after 12 mo but a strong osteolytic reaction after 24 mo. This was ascribed to the almost complete dissolution of α -TCP at this time and an adverse reaction of the remaining PDLLA.⁴⁸⁸

More complex calcium orthophosphate-based formulations are known as well. For example, there is a biocomposite consisting of three interpenetrating networks: TCP, CDHA and PLGA.⁴⁸⁹ First, a porous TCP network was produced by coating a polyurethane foam with a hydrolysable α-TCP slurry. Then, a CDHA network was derived from a calcium orthophosphate cement and used to fill in the porous TCP network. Finally, the remaining open pore network in the CDHA/ α-TCP structures was infiltrated with PLGA. This biocomposite consists of three phases with different degradation behaviors. It was postulated that bone would grow on the fastest degrading network of PLGA, while the remaining calcium orthophosphate phases would remain intact, thus maintaining their geometry and load-bearing capability.⁴⁸⁹

Formulations based on other calcium orthophosphates

The number of research publications devoted to formulations based on other calcium orthophosphates is substantially less than those devoted to apatites and TCP. Biphasic calcium phosphate (BCP), which is a solid composite of HA and β-TCP (however, similar formulations of HA and α-TCP, as well as of α-TCP and β-TCP, are possible as well) appears to be most popular among the remaining calcium orthophosphates. For example, collagen-coated BCP ceramics were studied, and their biocompatibility toward osteoblasts was found to increase upon coating with collagen.⁴⁹⁰ Another research group created porous PDLLA/BCP scaffolds and coated them with a hydrophilic PEG/vancomycin composite for both drug delivery purposes and surface modification.⁴⁹¹ More relevantly, both PLGA/BCP^492,493 and PLLA/BCP⁴⁹⁴ biocomposites were fabricated, and their cytotoxicity and fibroblast properties were found to be acceptable for natural bone tissue reparation, filling and augmentation.^495,496 PCL/BCP⁴⁹⁷ and gelatin/BCP^498,499 biocomposites are known as well.

A choice of DCPD-based biocomposites of DCPD, albumin and duplex DNA was prepared by a water/oil/water interfacial reaction method.³¹³ Core-shell-type DCPD/chitosan biocomposite fibers were prepared by a wet spinning method in another study.⁵⁰⁰ The energy-dispersive X-ray spectroscopy analysis indicated that Ca and P atoms were mainly distributed on the outer layer of the composite fibers; however, a small number of P atoms remained inside the fibers. This indicated that the composite fibers formed a unique core-shell structure with a shell of calcium orthophosphate and core of chitosan.⁵⁰⁰ A similar formulation was prepared for further applications in bone cement biocomposites.⁵⁰¹ DCPA/BSA biocomposites were synthesized through the coprecipitation of BSA on the nanodimensional particles of DCPA performed in ethanol.⁵⁰² Nanodimensional DCPA was synthesized and incorporated into dental resins to form dental biocomposites.^503-505 As an aside, it is interesting to mention that some DCPD/polymer composites could be used as proton conductors in battery devices.^506,507 Nothing has been reported on their biocompatibility yet, but perhaps sometime, improved formulations will be used to fabricate biocompatible batteries for implantable electronic devices.

Various ACP-based biocomposites and hybrid formulations for dental applications have been developed,^508-511 and several ACP-based formulations have been investigated as potential biocomposites for bone grafting^419,512-514 and drug delivery.⁵¹⁵ ACP/PPF biocomposites were prepared by in situ precipitation,⁵¹³ while PHB/carbonated ACP and PHBHV/carbonated ACP biocomposites appeared to be well-suited as slowly biodegradable bone substitution materials.⁴¹⁹ Another example is hybrid nanodimensional capsules, ~50–70 nm in diameter, which were fabricated by ACP mineralization of shell cross-linked polymer micelles and nano-sized cages.⁵¹⁴ These nano-sized capsules consisted of a continuous ultrathin inorganic surface layer that infiltrated the outer cross-linked polymeric domains. They might be used as structurally robust, pH-responsive biocompatible hybrid nanostructures for drug delivery, bioimaging and therapeutic applications.⁵¹⁴

Self-setting formulations and concretes

Inorganic self-setting calcium orthophosphate cements, which harden in the body, were introduced by LeGeros et al.⁵¹⁶ and Brown and Chow^517,518 in the early 1980s.⁵¹⁹ Since then, these cements have been broadly studied, and many formulations have been proposed. The cements set and harden due to various chemical interactions among calcium orthophosphates, which finally lead to formation of a monolithic body consisting of either CHDA or DCPD, with possible admixtures of other phases. Unfortunately, because of their ceramic natures, calcium orthophosphate cements are brittle after hardening, and the setting time is sometimes unsuitable for clinical procedures.⁵¹⁹ Therefore, various attempts have been made to transform the cements into biocomposites, e.g., by adding hydroxylcarboxylic acids to control the setting time,⁵²⁰ gelatin to improve both the mechanical properties and the setting time^473,521-523 or osteocalcin/collagen to increase the bioactivity.⁵²⁴ More to the point, various reinforcement additives of different shapes and nature are widely used to improve the mechanical properties of calcium orthophosphate cements. Even carbon nanotubes were used for this purpose!⁵²⁵ Although the biomaterials community does not use this term, a substantial amount of the reinforced cement formulations might be defined as calcium orthophosphate-based concretes.⁵²⁶ The idea behind the concretes is simple: if a strong filler is present in the matrix, it might stop crack propagation.

Various apatite-containing biocomposite formulations based on PMMA^527-540 and PEMA^102,541,542 have been already developed. Such biocomposites might be prepared by dispersion of apatite powder into a PMMA viscous fluid⁵⁴³ and could be used for drug delivery purposes.⁵⁴⁴ When the mechanical properties of the concretes composed of PMMA matrix and HA particles of various sizes were tested, the tensile results showed that strength was independent of particle size. In addition, up to 40% more weight in HA could be added without impairing the mechanical properties.^530,531 After immersion into Ringer’s solution, the tensile strength was not altered, whereas the fatigue properties were significantly reduced. The biocompatibility of PMMA/HA biocomposites was tested in vivo, and enhanced osteogenic properties of the implants compared with single-phase PMMA was observed.^528,532-535 It was shown that not only the mechanical properties of PMMA were improved, but the osteoblast response of PMMA was also enhanced with the addition of HA.⁵³² Thereby, by adding of calcium orthophosphates, a non-biodegradable PMMA was made more bioactive and osteoconductive, yielding a well-processible biocomposite concrete. As a drawback, the PMMA/HA formulations possess a low flexural, compressive and tensile strength.

A biocomposite made from HA granules and bis-phenol- α-glycidylmethacrylate-based resin appeared to possess comparable mechanical and biological properties to typical PMMA cement, leading to potential uses for implant fixation.⁵⁴⁵ To improve the mechanical properties of calcium orthophosphate cements and stabilize them at the implant site, various researchers have resorted to formulations that set in situ, primarily through cross-linking reactions of the polymeric matrix. For example, TTCP reacting with PAA formed a cross-linked CDHA/calcium polyacrylate biocomposite.⁵⁴⁶ In aqueous solutions, TTCP hydrolyzes to CDHA,²⁷ and the liberated calcium cations react with PAA, forming the cross-linked network.⁵⁴⁶ Reed et al. synthesized a dicarboxy polyphosphazene that can be cross-linked by calcium cations, and cement-based (TTCP + DCPD) CDHA/polyphosphazene biocomposites with a compressive strength ~10 MPa and of ~65% porosity were prepared as a result.⁵⁴⁷ To mimic PMMA cements, PFF/β-TCP biocomposites were prepared with the addition of vinyl monomer to cross-link PPF. As a result, quick-setting and degradable biocomposite cements with a low heat output and compressive strengths in the range of 1–12 MPa could be prepared by varying the molecular weight of PPF as well as the contents of the monomer, β-TCP initiator and NaCl as a porogen.^548,549 An acrylic cement with Sr-containing HA as a filler,¹³⁸ an injectable polydimethylsiloxane/HA cement,⁵⁵⁰ biocomposites consisting of PLGA microspheres and a calcium orthophosphate cement^551,552 as well as a hybrid cement formulation of chitosan oligosaccharide/gelatin/calcium orthophosphate⁵⁵³ were prepared as well.

In order to improve the mechanical properties of self-setting formulations, numerous researchers blended various polymers with the cements. For example, gelatin might be added to calcium orthophosphate cement formulations primarily to stabilize the paste in aqueous solution before it develops adequate rigidity and, second, to improve the compressive strength.^473,521,554 Adding rod-like fillers to the self-setting formulations also caused an improvement in the mechanical properties.⁵⁵⁴ For example, PAA and PVA were successfully used to improve the mechanical properties of a TTCP + DCPD cement but, unfortunately, with an inevitable and unacceptable reduction of both workability and setting time.^555,556 Similar findings were reported in the presence of sodium alginate and sodium polyacrylate.⁵⁵⁷ Other polymers, such as polyphosphazene, might be used instead.^558-560 Other examples of polymer/calcium orthophosphate cement formulations might be found elsewhere.^561,562

Porous calcium orthophosphate scaffolds with interconnected macropores (~1 mm), micropores (~5 µm) and of high porosity (~80%) were prepared by coating polyurethane foams with a TTCP + DCPA cement, followed by firing at 1,200°C. In order to improve the mechanical properties of the scaffolds, the open micropores of the struts were then infiltrated by a PLGA solution to achieve an interpenetrating bioactive ceramic/biodegradable polymer composite structure. The PLGA-filled struts were further coated with a 58S bioactive glass/PLGA composite coating. The complex porous biocomposites obtained could be used as tissue engineering scaffolds for low load-bearing applications.⁵⁶³ A more complicated construction, in which the PLGA macroporous phase has been reinforced with a bioresorbable TTCP + DCPA cement, followed by surface coating of the entire construct by a non-stoichiomentic CDHA layer, has been designed as well.⁵⁶⁴ The latter approach has culminated in a unique, three-phase biocomposite that is simple to fabricate, osteoconductive and completely biodegradable.

A porosity level of 42–80% was introduced into calcium orthophosphate cement/chitosan biocomposites by the addition of the water-soluble mannitol.⁵⁶⁵ Chitosan significantly improved the mechanical strength of the entire biocomposite.⁵⁶⁶ A similar approach was used by other researchers who studied the effect of the addition of PLGA microparticles^567-570 (which can also be loaded with drugs or growth factors^571-573) to calcium orthophosphate cements. These biocomposites were implanted into cranial defects of rats, and a content of ~30 wt% of the microparticles was found to give the best results,⁵⁶⁷ while the addition of a growth factor to the biocomposites significantly increased bone contact at 2 weeks and enhanced new bone formation at 8 weeks.⁵⁷³ The in vivo rabbit femur implant tests showed that PLGA/calcium orthophosphate cement formulations exhibited outstanding biocompatibility and bioactivity as well as a better osteoconduction and degradability than pure calcium orthophosphate cements.⁵⁶⁸

Formulations based on nanodimensional calcium orthophosphates and nanodimensional biocomposites

Nanodimensional and nanophasic materials are materials that have particles or grain sizes less than 100 nm, respectively. Thus, one should clearly differentiate between nanodimensional composites and composites based on nanodimensional compounds. The former might be any type of composite that has been disintegrated to particles with dimensions < 100 nm, while the latter are made up of two or more materials, in which at least one of the materials is of a nanometer scale.

Nanodimensional and nanophasic materials have different mechanical and optical properties than large grained materials of the same chemical composition. In particular, they possess unique surface properties, such as an increased number of atoms, grain boundaries and defects at the surface, huge surface area and altered electronic structure compared with conventional micron-sized materials. For example, nanodimensional HA (size ~67 nm) has a higher surface roughness of 17 nm compared with 10 nm for the conventional submicron size HA (~180 nm), while the contact angles (a quantitative measure of the wetting of a solid by a liquid) are significantly lower for nanodimensional HA (6.1) if compared with the conventional HA (11.51). Additionally, the diameter of individual pores in nanodimensional HA compacts is five times smaller (pore diameter ~6.6 Å) than that in the conventional grain-sized HA compacts (pore diameter within 19.8–31.0 Å).^574-576 Besides, nanodimensional HA promotes osteoblast cell adhesion, differentiation and proliferation, osteointegration and deposition of calcium-containing minerals on its surface better than microcrystalline HA, thus enhancing formation of a new bone tissue within a short period.^574-576 More to the point, nanodimensional HA was found to cause apoptosis of the leukemia P388 cells.⁵⁷⁷

Natural bones and teeth are hierarchical biocomposites of biological origin based on nanodimensional compounds, because they consist of nano-sized blade-like crystals of biological apatite grown in intimate contact with the organic matrix, which is rich in bioorganic fibers and organized in complicated hierarchical structures. Given the fact that the major bioorganic phase of bones is collagen, i.e., a natural polymer (Table 1), it is obvious that a composite of a nanodimensional calcium orthophosphate with a biodegradable polymer should be advantageous as bone substitution material. The inorganic nanodimensional phase would be responsible for the mechanical strength (hardness) and bioactivity, while the polymeric phase would provide the elasticity. In addition, the solubility of calcium orthophosphates depends on their crystallite size (smaller crystals have a higher solubility) and on their carbonate content (higher carbonate content increases the solubility).⁵⁷⁸ To the author’s best knowledge, among the calcium orthophosphates listed in Table 3, only apatites (CDHA, HA and, perhaps, FA) were available in a nanodimensional state until very recently. However, recently, nano-sized DCPA^503-505 and nano-sized MCPM⁵⁷⁹ have been synthesized and applied to prepare biocomposites with strong ionic release to combat tooth caries. Presumably, all the calcium orthophosphates in Table 3 might be manufactured in a nanodimensional and/or nanocrystalline state; however, not all of them have been prepared yet.

A number of investigations have been conducted recently to determine the mineralization, biocompatibility and mechanical properties of biocomposites based on various (bio)polymers and nanodimensional HA. Unfortunately, in the majority of the papers that have been published, it is unclear whether “nanodimensional HA,” in fact, represented the nanodimensional stoichiometric HA or a nanodimensional non-stoichiometric CDHA. These studies covered biocomposites with PLA^332,580-589 and its copolymer with PGA,^590-593 collagen,^594-607 collagen + PLA,^607-615 collagen + PVA,⁶¹⁶ collagen + alginate,^617,618 gelatin,^619-624 PPF,^625-627 polyamide,^{310,311,628-639} PVA,^{340,341,640-642} PVAP,³⁴⁵ poly(ethylene-co-acrylic) acid,^643,644 chitosan^645-651 and its derivatives,⁶⁵² konjac glucomannan + chitosan,⁶⁵³ PHEMA + PCL,⁶⁵⁴ PCL,^{390,435,655,656} cellulose,^{70,71,657-659} Ti,^660-662 PCL semi-interpenetrating biocomposites⁶⁶³ and many other biocompatible hybrid formulations.^{279,320,335,417,664-683} Furthermore, each of the aforementioned formulations might be covered by a layer of nanodimensional calcium orthophosphate, as was done by Zandi et al., who coated a biocomposite of nano-sized rods HA with gelatin by nano-sized HA. Several nanodimensional biocomposites were found to be applicable as carriers for delivery of drugs and growth factors^38,685-687 and were promising as vectors with ultra high gene loading and transfection efficiency.⁶⁸⁸ Data are available on the excellent biocompatibility of such biocomposites.⁶⁰⁵ The dispersion state of nano-sized particles appears to be the critical parameter in controlling the mechanical properties of nanodimensional biocomposites, as nano-sized particles always tend to aggregate owing to their high surface energy.⁴¹⁷ A comparison was made of the mechanical properties of biocomposites with nano-sized and micron-sized HA with a polyamide. The results showed that the bending and tensile strengths of the biocomposite increased with increasing content of nanodimensional HA but decreased with increasing micron-sized HA content.³¹⁰ A SEM image of the mineralized collagen fibrils demonstrating homogeneity of the nanodimensional biocomposite and the close interaction between the mineral phase and the reconstituted collagen fibrils is shown in Figure 4.⁶⁸⁹

Figure 4.

Scanning electron microscopy image of reconstituted mineralized collagen I fibrils. An example of an organic-inorganic nanostructural composite, mimicking the extracellular matrix of bone tissue on the nanometer scale. Reprinted from reference 689 with permission.

Porous (porosity ~85%) biocomposites of nano-sized HA with collagen and PLA have been prepared by precipitation and freeze-drying; these biocomposites did not show a pH drop upon in vitro degradation.^608-610 They were implanted in the radius of rabbits and showed a high biocompatibility and partial resorption after 12 weeks. Nano-sized HA/chitosan biocomposites with improved mechanical stability were prepared from HA/chitosan nano-sized rods.⁶⁹⁰ Nano-sized HA/PLLA biocomposites of high porosity (~90%) were prepared using thermally induced phase separation.⁶⁹¹ Nanodimensional HA was also used to prepare biocomposites with PAA, and the nanostructure of the resulting nano-sized crystals exhibited a core-shell configuration.^692,693

Nanodimensional crystals of HA appear to be suitable for intraosseous implantation and offer the potential to formulate enhanced biocomposites for clinical applications.⁶⁹⁴ For example, the biocompatibility of chitosan in osteoblast cell culture was significantly improved by the addition of nano-sized HA.⁶⁹⁵ Similar findings are valid for nanodimensional HA/polyamide biocomposites.⁶³⁰ Further details on nanodimensional biocomposites might be found in an excellent review in reference.36 More to the point, a more general review on applications of nanodimensional biomaterials in orthopedics is also available,⁶⁹⁶ where interested readers are referred.

Biocomposites with collagen

The main constituent of the bioorganic matrix of bones is type I collagen (Table 1) with molecules about 300 nm in length. The structural and biochemical properties of collagens have been widely investigated, and over 25 collagen subtypes have been identified.^697,698 This protein is conducive to crystal formation in the associated inorganic matrix. It is easily degraded and resorbed by the body and allows good attachment to cells. Collagen alone is not effective as an osteoinductive material, but it becomes osteoconductive in combination with calcium orthophosphates.⁶⁹⁹ Both collagen type I and HA were found to enhance osteoblast differentiation,⁷⁰⁰ but together, they were shown to accelerate osteogenesis. However, this tendency is not so straightforward: in the available data, implanted HA/collagen biocomposites enhanced regeneration of calvaria bone defects in young rats but postponed the regeneration of calvaria bone in aged rats.⁷⁰¹ Finally, the addition of calcium orthophosphates to collagen sheets was found to give a higher stability and an increased resistance to 3D swelling compared with collagen.⁷⁰² Therefore, a bone analog based on these two constituents should possess remarkable properties. Furthermore, addition of bone marrow constituents gives osteogenic and osteoinductive properties to calcium orthophosphate/collagen biocomposites.¹

The unique characteristics of bones originate from the spatial orientation between the nanodimentional crystals of biological apatite and collagen macromolecules at the nano scale,³⁹ where the crystals (about 50 nm length) are aligned parallel to the collagen fibrils,^25,26,35,42 which is believed to be the source of the mechanical strength of bones. The collagen molecules and the crystals of biological apatite assembled into mineralized fibrils are approximately 6 nm in diameter and 300 nm long.^{35,39,42,609,703} Although the complete mechanisms involved in the bone building strategy are still unclear, the strengthening effect of nanodimentional crystals of biological apatite in calcified tissues might be explained by the fact that the collagen matrix is a load-transfer medium and thus transfers the load to the intrinsically rigid inorganic crystals. Furthermore, the crystals of biological apatite located in between tangled fibrils cross-link the fibers, either through mechanical interlocking or by forming calcium ion bridges, thus increasing deformation resistance of the collagenous fiber network.⁷⁰⁴

When calcium orthophosphates are combined with collagen in a laboratory, the prepared biocomposites appear to be substantially different from natural bone tissue due to a lack of real interaction between the two components, i.e., the interactions that are able to modify the intrinsic characteristics of the singular components themselves. The main characteristic of the route by which the mineralized hard tissues are formed in vivo is that the organic matrix is laid down first, and the inorganic reinforcing phase grows within this organic matrix.^25,26,35,42 Although, to date, neither the elegance of the biomineral assembly mechanisms nor the intricate composite nano-sized architectures have been duplicated by nonbiological methods, the best way to mimic bone is to copy the way it is formed, namely by nucleation and growth of CDHA nano-sized crystals from a supersaturated solution both onto and within the collagen fibrils.^705-707 Such syntheses were denoted as “biologically inspired” which means they reproduce an ordered pattern and an environment very similar to natural ones.^708-710 The biologically inspired biocomposites of collagen and calcium orthophosphates (mainly, apatites) for bone substitute have a long history,^{33,442,597,711-730} which began with the pioneering study by Mittelmeier and Nizard,⁷³¹ who mixed calcium orthophosphate granules with a collagen web. Such combinations were found to be bioactive, osteoconductive, osteoinductive,^{33,699,732-734} and, in general, artificial grafts manufactured from this type of biocomposites are likely to behave similarly to bones and be of more use in surgery than those prepared from any other materials. Indeed, data are available on the superiority of calcium orthophosphate/collagen biocomposite scaffolds over the artificial polymeric and calcium orthophosphate bioceramic scaffolds individually.⁷³⁵

It has been found that calcium orthophosphates may be successfully precipitated onto a collagen substrate of whatever form or source.^{33,40,597,736,737} However, adherence of calcium orthophosphate crystals to collagen does depend on how much the collagen had been denatured: the more fibrillar the collagen, the greater attachment. Clarke et al. first reported the production of a biocomposite produced by precipitation of DCPD onto a collagen matrix with the aid of phosphorylated amino acids commonly associated with fracture sites.⁷¹⁶ Apatite cements (DCPD + TTCP) have been mixed with a collagen suspension, hydrated and allowed to set. CDHA crystals were found to nucleate on the collagen fibril network, producing a material with weaker mechanical properties than those reported for bone. Even more significantly, the prepared biocomposites were without a nanostructure similar to that of bone.^713,738 The oriented growth of OCP crystals on collagen was achieved by an experimental device in which Ca²⁺ and PO₄^3- ions diffused into a collagen disc from the opposite directions.^737,739,740 Unfortunately, these experiments were designed to simulate the mechanism of in vivo precipitation of biological apatite only; for this reason, the mechanical properties of the biocomposites were not tested.⁷⁴¹

Conventionally, collagen/calcium orthophosphate biocomposites have been prepared by blending or mixing of collagen and calcium orthophosphates, as well as by biomimetic methods.^{33,36,38,41,594,597,609,686,703,708-710,713,736,745-757} For example, Tampieri et al.⁷¹⁰ produced and compared artificial bone like tissue apatite/collagen biocomposites prepared using two different methodologies: (1) dispersion of apatite in a collagen aqueous suspension followed by freeze-drying and (2) direct nucleation of an apatitic phase on assembling collagen fibrils. Biocomposites obtained using first method were similar to uncalcified natural collagen. The crystallite sizes were not uniform and were often aggregated and randomly distributed into the matrix, proving that there was no real interaction between apatite and collagen fibers. However, the second method allowed the direct nucleation of nano-sized crystals of apatite on self-assembled collagen fibers. In this case, the two components (CDHA and collagen) exhibited strong interactions, highlighted by several analysis techniques, which showed a complete analogy of the composite with calcified natural tissue.⁷¹⁰ Other production techniques are also possible. For example, using a polymer-induced, liquid-precursor process, collagen/apatite biocomposites mimicking the nanostructure of bones, wherein nano-sized crystals of apatite were embedded within the collagen fibrils, were prepared.⁷⁵⁷ More complicated formulations, such as magnetite-enriched HA/collagen⁷⁵⁸ and HA/collagen/PVA⁷⁵⁹ biocomposites, have been developed as well.

Furthermore, collagen might be incorporated into various calcium orthophosphate cements.^{713,738,760-764} Typically, a type I collagen sponge is presoaked in a PO₄^3--containing, highly basic aqueous solution and then immersed into a Ca²⁺-containing solution to allow mineral deposition. Alternatively, collagen I fibers might be dissolved in acetic acid, then this solution added to phosphoric acid, followed by a neutralization synthesis (performed at 25°C and solution pH within 9–10) between an aqueous suspension of Ca(OH)₂ and the H₃PO₄/collagen solution.^708,709 To ensure the quality of the final product, it is necessary to control the Ca/P ionic ratio in the reaction solution. One way to do this is to dissolve a commercial calcium orthophosphate in an acid; another is to add Ca²⁺ and PO₄^3- ions in a certain ratio to the solution and, after that, induce the reaction.³⁹ Biomimetically, one can achieve an oriented growth of CDHA crystals onto dissolved collagen fibrils in aqueous solutions via a self-organization mechanism.⁷⁴⁷ A number of authors produced calcium orthophosphate/collagen biocomposites by mixing preformed ceramic particles with a collagen suspension.^765-767 However, in all blended composites, the crystallite sizes of calcium orthophosphates were not uniform, and the crystals were often aggregated and randomly distributed within a fibrous matrix of collagen. Therefore, no structural similarity to natural bone was obtained, and only a compositional similarity to that of natural bone was achieved. Instead, CDHA crystallization from aqueous solutions might be performed in the presence of a previously dispersed collagen,^33,597 or, more to the point, collagen might be first dispersed in an acidic solution, followed by addition of calcium and orthophosphate ions; then, coprecipitation of collagen and CDHA might be induced by either increasing the solution pH or adding mixing agents.⁴¹ Although it resulted in biocomposites with poor mechanical properties, pressing of the apatite/collagen mixtures at 40°C under 200 MPa for several days is also possible.⁷⁶⁸ Attempts have been made to create a computer simulation of the apatite/collagen composite formation process.⁷⁶⁹ It is interesting to note that such biocomposites were found to possess some piezoelectric properties.⁷⁷⁰

As the majority of the collagen/HA biocomposites are conventionally processed by anchoring micron-sized HA particles into a collagen matrix, it is quite difficult to obtain a uniform and homogeneous composite graft. Besides, such biocomposites have inadequate mechanical properties. Over and above, the proper pore sizes have not been achieved either. Further, microcrystalline HA, in contrast to nanocrystalline bone apatite, might take a longer time to be remodeled into a new bone tissue upon implantation. In addition, some of the biocomposites exhibit very poor mechanical properties, probably due to a lack of strong interfacial bonding between the constituents. The aforementioned data clearly demonstrate that a chemical composition similar to bone is insufficient for manufacturing the proper grafts; both the mechanical properties and mimetic bone nanostructure are necessary for a graft to function as bone in recipient sites. There is a chance for improving osteointegration by reducing the grain size of HA crystals by activating ultrafine apatite growth into the matrix. This may lead to the enhancement of mechanical properties and osteointegration, with improved biological and biochemical affinity to the host bone. Besides, porosity was found to have a positive influence on the ingrowth of the surrounding tissues into the pores of collagen/HA biocomposites.^771,772

Another approach is to mix bovine collagen with calcium orthophosphates. These biocomposites are marketed commercially as bone graft substitutes and can further be combined with bone marrow aspirated from the iliac crest of the site of the fracture. Collagraft^®, BioOss^® and Healos^® are several examples of the commercially available grafts for clinical use.³⁶ Application of these materials was compared with autografts for the management of acute fractures of long bones with defects that had been stabilized by internal or external fixation.^773,774 These biocomposites are osteogenic, osteoinductive and osteoconductive; however, they lack structural strength and require a harvest of the patient’s bone marrow. Although no transmission of diseases has been recorded yet, the use of bovine collagen might be a source of concern.²

Collagen sponges with an open porosity (30–100 µm) were prepared by a freeze-drying technique, and then their surface was coated by a 10 µm layer of biomimetic apatite precipitated from simulated body fluid.⁷⁷⁵ The researchers found a good in vitro performance with fibroblast cell culture. Other preparation techniques are also possible.⁷⁷⁶ Collagen/HA microspheres or gel beads have been prepared with the intention of making injectable bone fillers.^777,778 Liao et al. succeeded in mimicking the bone structure by blending carbonateapatite with collagen.⁷⁷⁹ A similar material (mineralized collagen) was implanted into femur of rats, and excellent clinical results were observed after 12 weeks.⁷⁸⁰ Collagen/HA biocomposites were prepared, and their mechanical performance was increased by cross-linking the collagen fibers with glutaraldehyde.^598,600,601 These biocomposites were tested in rabbits and showed a good biological performance, osteoconductivity and biodegradation. A similar approach was selected to prepare HA/collagen microspheres (diameter ~5 µm) by a water-oil emulsion technique in which the surface was also cross-linked by glutaraldehyde.⁷⁷⁸ That material showed a good in vitro performance with osteoblast cell culture. A porous bone graft substitute was formed from a nano-sized HA/collagen biocomposite combined with PLA by a freeze-drying method; the resulting material was found to mimic natural bones at several hierarchical levels.⁶⁰⁹ Subsequent in vitro experiments confirmed a good adhesion, proliferation and migration of osteoblasts into this composite.⁶⁰⁸ A further increase in biocompatibility might be achieved by the addition of various dopants. For example, to enhance bone substitution, Si-substituted HA/collagen composites have been developed, with silicon located preferentially in the collagen phase.⁵⁹⁹ Porous (porosity level ~95% with interconnected pores of 50–100 µm) biocomposites of collagen (cross-linked with glutaraldehyde) and β-TCP have been prepared by a freeze-drying technique, followed by sublimation of the solvent; the biocomposites showed a good biocompatibility upon implantation in the rabbit jaw.⁷⁸¹

Biocomposites of calcium orthophosphates with collagen were found to be useful for drug delivery purposes.^{618,721,764,782-784} Namely, an HA/collagen-alginate (20 µl) with rh-BMP2 (100 µg/ml, 15 µl) showed bone formation throughout the implant 5 weeks after implantation without obvious deformation of the material.⁶¹⁸ Gotterbarm et al. developed a two-layered collagen/β-TCP implant augmented with chondral inductive growth factors for repair of osteochondral defects in the trochlear groove of minipigs. This approach might be a promising new option for the treatment of deep osteochondral defects in joint surgery.⁷⁸³

To conclude this part, one should note that biocomposites of apatites with collagen are a very hot topic in research, and, up to now, just a few papers are devoted to biocomposites of other calcium orthophosphates with collagen.^{742-744,783,785-788} These biomaterials mimic natural bones to some extent, while their subsequent biological evaluation suggests that they are readily incorporated into the bone metabolism in a way similar to bone remodeling instead of acting as permanent implant.^609,731 However, the performance of these biocomposites depends on the source of the collagen from which it was processed. Several attempts have been made to simulate the collagen-HA interfacial behavior in real bone by means of cross-linking agents, such as glutaraldehyde,^{598,600,601,736,778,781} with the intention of improving the mechanical properties of these biocomposites. Unfortunately, further progress in this direction is restricted by high cost, the difficulty of controlling cross-infection, a poor definition of commercial sources of collagens as well as by a lack of appropriate technology for fabricating bone-resembling microstructures. Further details on calcium orthophosphate/collagen biocomposites might be found elsewhere.^36,725

Formulations with other bioorganic compounds and/or biological macromolecules

The biggest practical problems with collagen type I are its cost and the poor definition of commercial sources of this material, which makes it difficult to follow up on well-controlled processing. Therefore, collagen type I can be replaced by other compounds. One should notice that, besides collagen, both human and mammalian bodies contain dozens types of various bioorganic compounds, proteins and biological macromolecules. The substantial amount of them potentially might be used to prepare biocomposites with calcium orthophosphates. For example, a biologically strong adhesion (to prevent invasion of bacteria) between teeth and the surrounding epithelial tissues is attributed to a cell-adhesive protein, laminin.⁷⁸⁹ In order to mimic nature, a laminin/apatite biocomposite layer was successfully created on the surface of both titanium⁷⁹⁰ and EVOH^791,792 using the biomimetic approach. A more complicated laminin/DNA/apatite biocomposite layer was found to be an efficient gene transfer system.⁷⁹³

Calcium orthophosphate/gelatin biocomposites have been widely investigated as potential bone replacement biomaterials.^{317,336-338,444-452,466-474,487,521-523,554,619-624,794-807} For example, gelatin foams were successfully mechanically reinforced by HA and then cross-linked by a carbodiimide derivative.³¹⁷ Such foams were shown to be good carriers for antibiotic tetracycline.⁷⁹⁸ Several biocomposites of calcium orthophosphates with alginates have been prepared.^{471,617,618,622,709,808,809} For example, porous HA/alginate composites based on hydrogels were prepared both biomimetically⁷⁰⁹ and using a freeze-drying technique.⁸⁰⁸ Another research group succeeded in preparing biphasic but monolithic scaffolds using a similar preparation route.⁸¹⁰ Their biocompatibility in cell culture experiments and in vitro biodegradability were high; however, their mechanical strength could be better.

Various biocomposites of calcium orthophosphates with chitosan^{298,482,500,512,527,565,645-653,674,675,690,695,799,807,811-828} and chitin^{232,476,612,829-833} are also very popular. For example, a solution-based method was developed to combine HA powders with chitin, in which the ceramic particles were uniformly dispersed.^829,830 Unfortunately, it was difficult to obtain uniform dispersions. The mechanical properties of the final biocomposites were not very good; due to a poor adhesion between the filler and the matrix, both the tensile strength and modulus were found to decrease with increase of the amount of HA. Microscopic examination revealed that HA particles were intervened between the polymer chains, weakening their interactions and decreasing the overall strength.^829,830 Other manufacturing techniques might be found in the aforementioned references; I just would like to mention an interesting approach in which a HA/chitosan biocomposite was produced by a hydrothermal process from the natural CaCO₃/chitosan biocomposite of crab shells.⁸²⁷ Biocomposites of natural HA with chitosan were found to possess both a good hard tissue biocompatibility and an excellent osteoconductivity, which is suitable for artificial bone implants and frame materials for tissue engineering.⁸²³ Data are available that show the addition of HA into chitosan improved cell attachment and provided a higher cell proliferation and well-spread morphology when compared with chitosan alone.⁶⁵⁰ More complex formulations, such as silk fibers, reinforced HA/chitosan⁸³⁴ and HA/collagen/chitosan⁸³⁵ biocomposites, have been studied as well. Interestingly, hybrid biocomposites of nano-sized HA with chitin/chitosan might be used for removal of Fe(III)⁸³⁶ and fluorides^837,838 from aqueous solutions. Further details on the biocomposites and hybrid biomaterials of calcium orthophosphates with chitosan are available in the literature.⁸³⁶

Biocomposites of CDHA with water-soluble proteins, such as BSA, might be prepared by a precipitation method.^561,839-842 In such biocomposites, BSA is not strongly fixed to solid CDHA, which is useful for a sustained release. However, this is not the case if a water/oil/water interfacial reaction route has been used.³¹³ To extend this subject, inclusion of DNA into CDHA/BSA biocomposites was claimed.^313,843-845 Furthermore, nanodimentional biocomposites of an unspecified calcium orthophosphate with DNA⁸⁴⁶ as well as biocomposites of nano-sized crystals of biomimetic apatite with C₆₀ and Au-DNA nano-sized particles⁸⁴⁷ were also prepared.

Akashi and coworkers developed a procedure for preparing calcium orthophosphate-based biocomposites by soaking hydrogels in solutions supersaturated by Ca²⁺ and PO₄^3- ions in order to precipitate CDHA in the hydrogels (up to 70% more weight in CDHA could be added to these biocomposites).⁸⁴⁸ This procedure was applied to chitosan; the 3D shape of the resulting biocomposite was controlled by the shape of the starting chitosan hydrogel.⁸⁴⁹ Another research group developed biocomposites based on in situ calcium orthophosphate mineralization of self-assembled supramolecular hydrogels.⁸⁵⁰ Other experimental approaches are also possible.⁸⁵¹

Various biocomposites of CDHA with glutamic and aspartic amino acids as well as poly-glutamic and poly-aspartic amino acids have been prepared and investigated by Bigi et al.^{346,347,852-855} These (poly)amino acids were quantitatively incorporated into CDHA crystals, provoking a reduction of the coherent length of the crystalline domains and decreasing the crystal sizes. The relative amounts of the (poly)amino acid content in the solid phase, determined through HPLC analysis, increased with their concentration in solution up to a maximum of about 7.8 wt% for CDHA/aspartic acid and 4.3 wt% for CDHA/glutamic acid biocomposites. The small crystal dimensions, which implied a great surface area, and the presence of (poly)amino acids were suggested to be relevant for possible application of these biocomposites for hard tissues replacement.^{346,347,852-855} A schematic description of a biocomposite formation from amino acids and ACP is shown in Figure 5.⁸⁵⁶

Figure 5.

A proposed mechanism for the formation of ACP/amino acid biocomposites in aqueous solutions. Reprinted from reference 856 with permission.

Furthermore, BCP (HA + β-TCP)/agarose macroporous scaffolds with controlled and complete interconnection, high porosity, thoroughly open pores and tailored pore size were prepared for tissue engineering applications.^857,858 Agarose, a biodegradable polymer, was selected as the organic matrix, because it is a biocompatible hydrogel that acts as gelling agent, leading to strong gels and fast room temperature polymerization. Porous scaffolds with the designed architecture were manufactured by combining a low temperature shaping method with stereo-lithography and two drying techniques. The biocompatibility of this BCP/agarose system was tested with mouse L929 fibroblasts and human SAOS-2 osteoblasts during different colonization times.⁸⁵⁹

Fibrin sealants are non-cytotoxic, fully resorbable biological matrices that simulate the last stages of a natural coagulation cascade, forming a structured fibrin clot similar to a physiological clot.⁸⁶⁰ Biocomposites of calcium orthophosphates with fibrin sealants might help develop the clinical applications of bone substitutes. The 3D mesh of fibrin sealant interpenetrates the macro- and microporous structure of calcium orthophosphate ceramics.¹¹ The physical, chemical and biological properties of calcium orthophosphate bioceramics and the fibrin glue might cumulate in biocomposites suitable for preparation of advanced bone grafts.^861-873

Furthermore, there are biocomposites of calcium orthophosphates with bisphosphonates,⁸⁷⁴ silk fibroin (that is a hard protein extracted from silk cocoon),^{312,670-672,677,678,875-881} chitosan + silk fibroin,⁸⁸² fibronectin,⁸⁸³ chondroitin sulfate,^299,733,884 casein phosphopeptides⁸⁸⁵ and vitamins.⁸⁸⁶ The reader’s attention is also drawn to an interesting approach to crystallizing CDHA inside poly(allylamine)/poly(styrene sulfonate) polyelectrolyte capsules, resulting in empty biocomposite spheres of micron size.⁸⁸⁷ Depending on the amount of precipitated CDHA, the thickness of the shell of biocomposite spheres can be varied between 25 and 150 nm. These biocomposite capsules might find application as medical agents for bone repairing and catalytic microreactors.⁸⁸⁷

Injectable bone substitutes (IBS)

With the development of minimally invasive surgical methods, for example, percutaneous surgery, directly injectable biomaterials are needed. The challenge is to place a biomaterial at the site of surgery by the least invasive method. In this regard, IBS appear to be a convenient alternative to solid bone-filling materials. They represent ready-to-use suspensions of calcium orthophosphate microspheres,^888,889 nano-sized rods⁸⁹⁰ or powder(s) in a liquid carrier phase. However, addition of other phases, such as calcium sulfate,⁸⁹¹ is also possible. They look like opaque viscous pastes with rheological properties sufficient to inject them into bone defects by means of surgical syringes and needles. Besides, IBS could be easily produced in a sterile stage. Their stable composition and mechanical properties are suitable for reproducibility of the biological response.⁸⁹² All types of IBS are divided into two major groups: self-setting formulations and those that do not set. Cements and concretes belong to the former (see the “Self-setting formulations and concretes” section above), while those that fall into the latter are described here.

IBS requires suitable rheological properties to ensure bonding of the mineral phase in situ with good cell permeability. Usually, the necessary level of viscosity is created by addition of water-soluble polymers.^131,893,894 Therefore, the majority of calcium orthophosphate-based IBS formulations might be considered a subgroup of calcium orthophosphate/polymer biocomposites. For example, an IBS was described that involved a silanized hydroxyethylcellulose carrier with BCP (HA + β-TCP).⁸⁹⁵ The suspension is liquid at a pH within 10–12, but it gels quickly at a pH < 9. Injectable composites can be formed with β-TCP to improve mechanical integrity.⁵⁴⁸ Similarly, Bennett et al. showed that a polydioxanone-co-glycolide-based biocomposite reinforced with HA or β-TCP can be used as an injectable or moldable putty.⁸⁹⁶ During the cross-linking reaction following injection, carbon dioxide is released, allowing the formation of interconnected pores. Furthermore, HA/poly(L-lactide-co-Σ-caprolactone) biocomposite microparticles were fabricated as an injectable scaffold via the Pickering emulsion route in the absence of any molecular surfactants. A stable injectable oil-in-water emulsion was obtained using water-dispersed HA nano-sized crystals as the particulate emulsifier and a dichloromethane solution of poly(L-lactide-co-Σ-caprolactone) as an oil phase.⁸⁹⁷

Daculsi et al. developed viscous IBS biocomposites based on BCP (60% HA + 40% β-TCP) and a 2% aqueous solution of HPMC that was said to be perfectly biocompatible, resorbable and easily fitted to bone defects (due to an initial plasticity).^{108,894,898-905} The best ratio BCP/HPMC aqueous solution was found to be at ~65/35 w/w. To extend this subject further, IBS might be loaded by cells,^906,907 radiopaque elements⁹⁰⁸ or microparticles⁹⁰⁹ as well as be functionalized by nucleic acids.⁸⁹⁰ Self-hardening formulations based on Si-HPMC hydrogel are known as well.⁹⁰⁶ The list of the commercially available calcium orthophosphate-based IBS formulations is presented in Table 5.⁹¹⁰

Table 5. A list of some commercial non-setting calcium orthophosphate IBS and pastes with indication of producer, product name, composition (when available) and form.⁹¹⁰ .

Producer	Product name	Composition	Form
ApaTech (UK)	Actifuse™	HA, polymer and aqueous solution	pre-mixed
	Actifuse™ Shape Actifuse™ ABX	Si-substituted calcium orthophosphate and a polymer	pre-mixed
Baxter (US)	TricOs Τ TricOs	BCP (60% HA, 40% β-TCP) granules and Tissucol (fibrin glue)	to be mixed
Berkeley Advanced Biomaterials	Bi-Ostetic Putty	not disclosed	not disclosed
BioForm (US)	Calcium hydroxylapatite implant	HA powder embedded in a mixture of glycerine, water and carboxymethylcellulose	pre-mixed
Biomatlante (FR)	MBCP Gel®	BCP granules (60% HA, 40% β-TCP; 0.08 – 0.2 mm) and 2% HPMC	pre-mixed
	Hydr’Os	BCP granules (60% HA, 40% β-TCP; micro- and nano-sized particles) and saline solution	pre-mixed
Degradable solutions (CH)	Easy graft™	β-TCP or BCP granules (0.45 – l.0 mm) coated with 10 μm PLGA, N-methyl-2-pyrrolydone	to be mixed
Dentsply (US)	Pepgen P-15® flow	HA (0.25 – 0.42 mm), P-15 peptide and aqueous Na hyaluronate solution	to be mixed
DePuy Spine (US)	Healos® Fx	HA (20 – 30%) and collagen	to be mixed
Fluidinova (P)	nanoXIM TCP	β-TCP (5 or 15%) and water	pre-mixed
	nanoXIM HA	HA (5, 15, 30 or 40%) and water	pre-mixed
Integra LifeSciences (US)	Mozaik Osteoconductive Scaffold	β-TCP (80%) and type 1 collagen (20%)	to be mixed
Mathys Ltd (CH)	Ceros® Putty / cyclOS® Putty	β-TCP granules (0.125 – 0.71 mm; 94%) and recombinant Na hyaluronate powder (6%)	to be mixed
Medtronic (US)	Mastergraft®	BCP (85% HA, 15% β-TCP) and bovine collagen	to be mixed
Osartis / ΑΑΡ (GER)	Ostim®	Nanocrystalline HA (35%) and water (65%)	pre-mixed
Smith and Nephew (US)	JAXTCP	β-TCP granules and an aqueous solution of 1.75% carboxymethylcellulose and 10% glycerol	to be mixed
Stryker (US)	Calstrux™	β-TCP granules and carboxymethylcellulose	to be mixed
Teknimed (FR)	Nanogel	HA (100 – 200 nm) (30%) and water (70%)	pre-mixed
Therics (US)	Therigraft™ Putty	β-TCP granules and polymer	pre-mixed
Zimmer (US)	Collagraft	BCP granules (65% HA, 35% β-TCP; 0.5 – 1.0 mm), bovine collagen and bone marrow aspirate	to be mixed

The advanced characteristics of IBS come from their good rheological properties and biocompatibility and the ease of tissue regeneration. Although the fabrication of IBS biocomposites, in most cases, improved the mechanical properties of the system and provided the material with resistance to fluids penetration, these achievements were limited by the amount of polymer that can be added to the paste. For instance, Mickiewicz et al. reported that after a critical concentration (which depended on the type and molecular weight of the polymer but was always around 10%), the polymer started forming a thick coating on the crystal clusters, preventing them from interlocking, originating plastic flow and, as a consequence, decreasing their mechanical properties.⁵⁶¹ More to the point, Fujishiro et al. reported a decrease in mechanical properties when using higher amounts of gel, which was attributed to formation of pores due to leaching of gelatin in solution.⁵⁵⁴ Therefore, it seems that mechanical properties, although improved by the addition of polymers, are still a limitation for the application of calcium orthophosphate-based IBS formulations in load-bearing sites.¹⁷⁸ Further details on IBS might be found in a recent review in reference.892

Biocomposites with glasses, inorganic materials, carbon and metals

To overcome the problem of poor mechanical properties of calcium orthophosphate bioceramics, suitable biocomposites of calcium orthophosphates reinforced by various inorganic materials, glasses and metals have been developed. Such biocomposites are mainly prepared by common ceramic processing techniques, such as thermal treatment after kneading,^911-913 powder slurry coating⁹¹⁴ and metal-sol mixing.⁹¹⁵ For example, HA was combined with Bioglass^® (Novabone Products, Alachua, FL)^916,917 and with other glasses⁹¹⁸ to form glass-ceramic biocomposites. Other reinforcement materials for calcium orthophosphates are differentiated either by shape of the fillers, namely, particles,^919,920 platelets,^921,922 whiskers,^579,923-925 fibers^926-930 or by their chemical composition, zirconia and/or PSZ,^{313,911-914,923,931-966} alumina,^{313,919,922,965,967-996} other oxides,^{925,997–1004} silica and/or glasses,^1005–1014 wollastonite,^{206,1015–1025} mullite,^1026,1027 various metals and alloys,^{540,928,967,997,1028–1045} calcium sulfate,^1046–1049 calcium carbonate,^1050,1051 silicon carbide,^683,924 barium titanate,¹⁰⁵² zeolite,¹⁰⁵³ boron nitride¹⁰⁵⁴ and several other materials.^{335,1055–1057} More complicated formulations, such as HA/aluminum oxide/carbon nanotubes,¹⁰⁵⁸ have been developed as well. All these materials have been added to calcium orthophosphate bioceramics to improve their reliability. Unfortunately, significant amounts of the reinforcing phases are needed to achieve the desired properties and, as these materials are either bioinert, significantly less bioactive than calcium orthophosphates or not bioresorbable, the ability of the biocomposites to form a stable interface with bone is poor compared with calcium orthophosphate bioceramics alone. Due to the presence of bioinert compounds, such formulations might be called bioinert/bioactive composites.¹⁰⁰⁵ The ideal reinforcement material would impart mechanical integrity to a biocomposite at low loadings without diminishing its bioactivity.

There are several types of HA/glass biocomposites. The first one is also called bioactive glass-ceramics. A dense and homogeneous biocomposite was obtained after a heat treatment of the parent glass, which comprised ~38 wt% oxy-FAP (Ca₁₀(PO₄)₆(O,F)₂) and ~34 wt% β-wollastonite (CaO·SiO₂) crystals, 50–100 nm in size in a MgO-CaO-SiO₂ glassy matrix.^{206,1015–1025} A-W glass-ceramics are an assembly of small apatite particles effectively reinforced by wollastonite. The bending strength, fracture toughness and Young’s modulus of A-W glass-ceramics are the highest among bioactive glass and glass-ceramics, enabling them to be used in some major compression load-bearing applications, such as vertebral prostheses and iliac crest replacement. They combine a high bioactivity with suitable mechanical properties.¹⁰⁵⁹ β-TCP/wollastonite biocomposites are also known.^1060–1062 More complicated formulations have been developed as well. For example, (A-W)/HDPE composite (AWPEX) biomaterials have been designed to match the mechanical strength of human cortical bone and to provide favorable bioactivity, with potential use in many orthopedic applications.^1063–1066 Other examples include wollastonite-reinforced HA/Ca polycarboxylate,¹⁰⁶⁷ glass-reinforced HA/polyacrylate¹⁰⁶⁸ as well as collagen¹⁰⁶⁹ and gelatin¹⁰⁷⁰ calcium phosphate silicate/wollastonite biocomposites.

HA/glass biocomposites can be prepared by simple sintering of appropriate HA/glass powder mixtures.^1071–1074 If sintering is performed below 1,000°C, HA does not react with the bioactive glass^1072,1073 or the reaction is limited.¹⁰⁷⁴ Besides, reactions between HA and glasses depend on the glass composition. In another approach, small quantities of bioactive glass have been added to HA bioceramics in order to improve densification and/ or mechanical properties.²⁹ Biocomposites might also be sintered from HA and silica.¹⁰⁰⁵ In general, bioactive glass-ceramics maintain a high strength for a longer time than HA bioceramics under both in vitro and in vivo conditions.^1012,1019

Due to a huge difference in shapes, it is a challenge to prepare homogeneous mixtures of calcium orthophosphates and carbon nanotubes: “one can imagine something similar to achieving a homogeneous mixture of peas and spaghetti.”²¹⁷ Nevertheless, different strategies might be employed to prepare calcium orthophosphate/carbon nanotube biocomposites. For example, apatites might be chemically synthesized by using carboxyl functionalized carbon nanotubes as a matrix.^302-308 Physicochemical characterization of these biocomposites showed that nucleation of CDHA is initiated through the carboxyl group.³⁰² Hot pressing,¹⁰⁷⁵ plasma spraying,¹⁰⁷⁶ laser surface alloying,^1077–1079 spark plasma sintering¹⁰⁸⁰ and precipitation¹⁰⁸¹ techniques might be applied as well. Due to carbon oxidation at elevated temperature, sintering of calcium orthophosphate/carbon nanotube biocomposites must be performed in a deoxidizing atmosphere.¹⁰⁸² The research on calcium orthophosphate (up to now, only apatites)/carbon nanotube biocomposites is in its early stages, with the first papers published in 2004.^307,525 For this reason, the mechanical property data for such biocomposites have been reported only in a few papers; however, these results are encouraging. For example, Chen et al. performed nano-indentation tests on biocomposite coatings to give hardness and Young’s modulus values.¹⁰⁷⁹ They found that the higher the loading of the nanotubes, the better the mechanical properties. Namely, at 20 wt% loading, hardness was increased by ~43% and Young’s modulus by ~21% over a single-phase HA coating.¹⁰⁷⁹ Scratching test results indicated that alloyed HA biocomposite coatings exhibited improved wear resistance and a lower friction coefficient when the amount of carbon nanotubes in the precursor material powders was increased.¹⁰⁷⁸ Additionally, measurements of the elastic modulus and hardness of the biocomposite coatings indicated that the mechanical properties were also affected by the amount of carbon nanotubes.¹⁰⁷⁷ Another research group performed compression tests on bulk HA/carbon nanotubes biocomposites and found an increase in strength over single-phase HA.³⁰⁷ However, the highest compressive strength they achieved for any material was only 102 MPa, which is similar to that of cortical bone but much lower than the typical values for dense HA.²¹⁷ More complex formulations, such as poly-l-lysine/HA/carbon nanotube hybrid biocomposites, have also been developed.¹⁰⁸³ Furthermore, calcium orthophosphate/carbon nanotube biocomposites might be immobilized by hemoglobin.¹⁰⁸⁴ Unfortunately, carbon nanotubes are very stable substances; they are neither bioresorbable nor biodegradable. Therefore, during in vivo bioresorption, the nanotubes will get into the human body from the biocomposite matrix and might cause uncertain health problems. Certainly, this problem must be solved. To conclude the carbon subject, one should mention the application of carbon fibers of microscopic dimensions,^1085–1087 nanodimensional diamonds¹⁰⁸⁸ and C₆₀⁸⁴⁷ to reinforce HA bioceramics.

As clearly seen from the amount of references, apatite/zirconia biocomposites are the most popular among researchers. The main disadvantage of HA reinforced by PSZ is degradation of zirconia in wet environments.^{923,932,933,955} Transformation of the tetragonal ZrO₂ to the monoclinic phase on the surface results in the formation of microcracks and, consequently, lowers the strength of the implant.^1089,1090 Interestingly, though, Fe₃O₄/HA composites possess photocatalytic properties.^1003,1004

Various biocomposites of calcium orthophosphates with metals and alloys have been fabricated as well.^{540,928,967,997,1028–1045} For example, an HA-based biocomposite reinforced with 20 vol.% of Ti particles was fabricated by hot pressing.¹⁰³⁰ Calcium orthophosphate/Ti biocomposites might be prepared by powder metallurgy processing.^1032–1034 At high temperatures, the presence of Ti metal phase was found to promote dehydration and decomposition of HA into β-TCP and TTCP^1030,1032 or partial formation of β-TCP and calcium titanate instead of HA.^{661,1033,1034} Compared with pure HA bioceramics manufactured under the same conditions, the HA/Ti biocomposites possessed a higher fracture toughness, bending strength, work of fracture, porosity and lower elastic modulus, which makes them more suitable for biomedical applications. However, the mechanical properties appear not to be high enough to use HA/Ti biocomposites in load-bearing applications. Luckily, the histological evaluations revealed that HA/Ti biocomposites could be partially integrated with newborn bone tissues after 3 weeks and fully osteointegrated at 12 weeks in vivo.¹⁰³⁰ Similar findings had earlier been made for HA bioceramics reinforced by the addition of silver particulates (5–30 vol. %) and subsequent sintering of the HA/Ag powder compacts.^1028,1029 The addition of silver also imparts an antimicrobial activity.¹⁰⁴² Other studies on calcium orthophosphate/Ti biocomposites are available elsewhere in reference 1035–1038.

To conclude this section, biocomposites consisting only of calcium orthophosphates should be briefly described. First, all multiphasic and polyphasic calcium orthophosphates should be mentioned. For example, ca. 1980, BCP was described as “TCP ceramics complexed with HA,”¹⁰⁹¹ Even nowadays BCP is occasionally called a “nanocomposite,”¹⁰⁹² Furthermore, fluoridated HA [described by a chemical formula Ca₁₀(PO₄)₆(OH)_{2 - x}F_x, where 0 < x < 2] might be mentioned as a composite;¹⁰⁹³ however, the applicability of the term “composite” for such systems is doubtful. One should better consider 70% HA-powder + 30% HA-whisker biocomposites, which were fabricated by pressureless sintering, hot pressing and hot isostatic pressing. These biocomposites were found to exhibit an improved toughness, attaining the lower fracture toughness limit of bone without a decrease of bioactivity and biocompatibility.^1094,1095 A dual HA biocomposite that combined two HA materials with different porosities: HA with 75% porosity, for bone ingrowth, and HA with 0% porosity, for load bearing, was also manufactured. This dual HA biocomposite appeared to be suitable for use as an implant material for spinal interbody fusion as a substitute for iliac bone grafts, which could eliminate the disadvantages associated with autograft harvesting.¹⁰⁹⁶ A biodegradable biocomposite porous scaffold comprised of a β-TCP matrix and nano-sized fibers of HA was developed and studied for load-bearing bone tissue engineering. The nano-sized fibers of HA were prepared by a biomimetic precipitation method, the inclusion of which significantly enhanced the mechanical property of the scaffold, attaining a compressive strength of 9.87 MPa, comparable to the high-end value (2–10 MPa) of cancellous bone.¹⁰⁹⁷ Finally, it is interesting to mention a successful reinforcement of carbonateapatite porous blocks by newly prepared carbonateapatite crystals (i.e., by the same compound; thus, a biocomposite of two different carbonateapatites was obtained).¹⁰⁹⁸ First, a calcium salt was introduced to micropores of carbonateapatite blocks. Then, the calcium salt was carbonated to form calcite inside the micropores of the carbonateapatite blocks by exposing the blocks to carbon dioxide. For the third step, the blocks were immersed in a Na₂HPO₄ aqueous solution. In this process, calcite inside the micropores of the carbonateapatite blocks was transformed to carbonateapatite and the newly formed crystals of carbonateapatite entangled on those of the existing carbonateapatite blocks. Due to bonding between the newly formed carbonateapatite crystals and the existing ones in the carbonateapatite blocks, the mechanical strength of the blocks became ~1.5 times higher compared with that before the treatment.¹⁰⁹⁸

Functionally graded formulations

Although, in most cases, the homogeneous distribution of filler(s) inside a matrix is required,⁴²⁶ there are composites where this is not the case. For example, functionally graded materials (commonly referred to as FGM) might be characterized by the intentional variations in composition and/or structured gradually over volume, resulting in corresponding changes in the properties of the composite. The main feature of such materials is the almost continuously graded composition, which results in two different properties at the two ends of the structure. Such composites can be designed for specific functions and applications. Various approaches based on bulk (particulate) processing, preform processing, layer processing and melt processing are used to fabricate the functionally graded materials.

Bone is a biologically formed composite with variable density ranging from very dense and stiff (cortical bone) to a soft and foamed structure (trabecular bone). Normally the outer part of long bones consists of cortical bone, with the density decreasing toward the core, where the trabecular bone is found. The trabecular bone is porous, and the pores are filled with osseous medulla.^25,26 This brief description clearly indicates that bones are natural functionally graded composites.

The concept of FGM has been increasingly used for biomaterial design and, currently, it remains an important area of research. For example, many studies have been performed to fabricate porosity-graded calcium orthophosphate bioceramics in attempts to mimic the porous structure of bones.^1099–1102 This is a structural approach to fabricating FGM. Besides that, there is a compositional approach. For example, powder metallurgy methods have been used to fabricate HA/Ti functionally graded biocomposite dental implants, offering the biocompatible HA on the tissue side and titanium on the outer side for mechanical strength.^1103–1105 The graded structure in the longitudinal direction contains more Ti in the upper section and more HA in the lower section. Actually, in the upper section, the occlusal force is directly applied, and Ti offers the required mechanical performance; in the lower part, which is implanted inside the bone, the HA confers bioactive and osteoconductive properties to the material.¹¹⁰³ Since the optimum conditions of sintering for Ti and HA are very different, HA/Ti functionally graded biocomposites are difficult to fabricate, and the sintering conditions for their mixtures are obliged to compromise. The expected properties of this implant are shown in Figure 6.¹¹⁰⁴ Such biocomposites might be both symmetrical¹¹⁰⁶ and asymmetrical.¹¹⁰⁷ Furthermore, functionally graded HA/Ti biocomposite coatings might be prepared by RF plasma spraying.¹¹⁰⁸ More to the point, a Ti alloy substrate has been combined with HA granules that could be spread over the surface.¹¹⁰⁹

Figure 6.

Expected properties of functionally graded biocomposite dental implant. For comparison, the upper drawing shows a functionally graded implant and the lower one shows a conventional uniform implant. The properties are shown in the middle. The implant with the composition changed from a biocompatible metal (Ti) at one end (left in the figure), increasing the concentration of bioceramics (HA) toward 100% HA at the other end (right in the figure), could control both mechanical properties and biocompatibility without an abrupt change due to the formation of discrete boundary. This FGM biocomposite was designed to provide more titanium for the upper part where occlusal force is directly applied and more HA for the lower part, which is implanted inside the jawbone. Reprinted from reference 1104 with permission.

A series of functionally graded HA coatings incorporated with various percentages of silver were deposited on titanium substrates using ion beam-assisted deposition. The analysis of the coating’s cross-section revealed a decreased crystallinity as well as a distribution of nano-sized (10–50 nm) silver particles from the coating/substrate interface to top surface.¹¹¹⁰ A functionally graded HA/PMMA biocomposite was developed based on sedimentary HA distributions in a PMMA viscous fluid, using a centrifuge to avoid stress convergence on the interface. The stress-strain curves of this biocomposite showed sufficient strength for biomedical applications along with the relaxation of brittleness and fragility.⁵⁴³ A compositionally graded collagen/nanodimensional HA biocomposite scaffold might be prepared by an in situ diffusion method.¹¹¹¹ Chemical and microstructural analysis revealed a gradient of the Ca-to-P ratio across the width of the scaffold template, resulting in the formation of a Ca-rich side and a Ca-depleted side of the scaffold. The Ca-rich side featured low porosity and agglomerates of the nanodimensional HA crystallites, while the Ca-depleted side featured higher porosity and nanodimensional HA crystallites integrated with collagen fibrils to form a porous network structure.¹¹¹¹ A three-layered, graded biocomposite membrane with one face of 8% nanodimensional carbonateapatite/collagen/PLGA porous membrane, the opposite face of pure PLGA non-porous membrane and the middle layer of 4% nanodimensional carbonateapatite/collagen/PLGA as the transition was prepared using the layer-by-layer casting method.⁶¹¹ Functionally graded non-woven meshes of PCL incorporated by nano-sized particles of β-TCP were prepared using a hybrid twin-screw extrusion/electrospinning process.¹¹¹² A functionally graded HA/silk fibroin biocomposite was prepared by pulse electric current sintering.¹¹¹³ HA/glass FGM layers were coated on titanium alloy (Ti-6Al-4V) substrates. The design of these layers and the use of the glass were meant to achieve a strong bond between the FGM layered coatings and the substrates.^1114,1115

Functionally graded β-TCP/FA biocomposites combine the biostability of FA with the bioresorbable properties of β-TCP.¹¹¹⁶ An interesting multilayered (each layer 1 mm thick) structure consisting of β-TCP/FA biocomposites with different molar ratios has been prepared, giving rise to formation of an FGM (Fig. 7). After implantation, the preferential dissolution of the β-TCP phase would result in functionally gradient porosity for bone ingrowth.¹¹¹⁶ Functionally graded fluoridated HA with a gradient of fluoride¹¹¹⁷ and carbonated HA with a gradient of carbonate¹¹¹⁸ were synthesized as well. HA/zirconia graded biocomposites were fabricated to enhance the mechanical properties of HA while retaining its bone bonding property.⁹⁶³ TiO₂ and HA were found to be a good combination for FGM, providing both a gradient of bioactivity and good mechanical strength.¹¹¹⁹ In addition, graded HA/CaCO₃ biocomposite structures for bone ingrowth were also developed.¹¹²⁰ Functionally graded composite skull implants consisting of polylactides, carbonateapatite and CaCO₃ are known as well.^386,387 Thus, the research in this field is quite promising, but currently, the mechanical properties of the available biocomposites do not match the corresponding properties of bones.¹⁷⁹

Figure 7.

A schematic diagram showing the arrangement of the FA/β-TCP biocomposite layers. (A) A non-symmetric functionally gradient material (FGM); (B) symmetric FGM. Reprinted from reference 1116 with permission.

Biosensors

A biosensor is a device for detection of an analyte that combines a biological component with a physicochemical detector component. Very briefly, it consists of three parts: a sensitive biological element, a transducer or a detector element, which transforms the signal resulting from the interaction of the analyte and the biological element into another signal, and the associated electronics, which are primarily responsible for the display of the results in a user-friendly way.¹¹²¹

The surface of biologically relevant calcium orthophosphates (CDHA, HA, α-TCP, β-TCP, DCPD, DCPA) has an excellent capacity for adsorption of functional biomolecules, such as proteins, albumins, DNA as well as some other types of chemicals. Therefore, several calcium orthophosphate-based biocomposites and hybrid biomaterials were found to be applicable for biosensor manufacturing.^{354,642,1041,1083,1122–1127} For example, formation of poly-l-lysine/HA/carbon nanotube hybrid nanodimensional particles was described, and a general design strategy for an immunosensing platform was proposed based on adsorption of antibodies onto this biocomposite.¹⁰⁸³ In another paper, a hybrid material formed by assembling nanodimensional particles of gold onto nano-sized HA was employed for the interface design of a piezoelectric immunosensor on which the antibodies were bound. The sensing interface that was developed appeared to possess some advantages, such as activation-free immobilization and high antigen-binding activities of antibodies, over using nano-sized either HA or gold alone.¹⁰⁴¹ A novel tyrosinase biosensor based on nano-sized HA/chitosan composites has been developed for the detection of phenolic compounds.¹¹²⁵ Further details on the subject are available in the aforementioned references.

To date, not many papers have been published on the biosensor application of calcium orthophosphate-based biocomposites and hybrid biomaterials. Presumably, this subject will be further developed in future, and, perhaps sometime, implantable biosensors will be designed to perform the continuous concentration monitoring of the important biological macromolecules in vivo. Possibly, those implantable biocencors will be able to use electric power generated by DCPD/polymer composite-based battery devices.^506,507

Interaction Among the Phases in Calcium Orthophosphate-Based Formulations

An important aspect that should be addressed in detail is a mutual interaction among calcium orthophosphates and other phases in biocomposites and hybrid biomaterials. In general, an interaction among the phases in any composite can be either mechanical, when it results from radial compression forces exerted by the matrix on the filler particles (for example, developed during cooling due to thermal contraction), or chemical, when the reactivity of the filler toward the matrix has an important role. In the latter case, it is important to distinguish a physical interaction from chemical bonding.²⁸² According to Wypych,¹¹²⁸ physical interaction is more or less temporary, implicating hydrogen bonding or van der Waals forces, whereas chemical bonding is stronger and more permanent, involving covalent bond formation. Thus, a chemical interfacial bond among the phases is preferred to achieve higher strength in a composite. The magnitude of the interfacial bond among the phases determines how well a weak matrix transmits stress to the strong fibers. However, while a bond among the matrix and reinforcements must exist for the purpose of stress transfer, it should not be so strong that it prevents toughening mechanisms, such as debonding and fiber pullout.²¹⁷

There is still doubt as to the exact bonding mechanism among bone minerals (biological apatite) and bioorganics (collagen), which undoubtedly plays a critical role in determining the mechanical properties of bones. Namely, bone minerals are not bonded directly to collagen but through non-collagenous proteins that make up ~3% of bones (Table 1) and provide with active sites for biomineralization and for cellular attachment.³⁶ In bones, the interfacial bonding forces are mainly ionic bonds, hydrogen bonds and hydrophobic interactions, which give the bones their unique composite behavior.⁵³ There is an opinion that, in contrast to bones, there is no sign of chemical bonding among the phases in conventional calcium orthophosphate/collagen biocomposites, probably due to a lack of suitable interfacial bonding during mixing.³⁹ However, this is not the case for phosphorylated collagens.⁷⁵³ Interested readers are directed to a density functional theory study of the interaction of collagen peptides with hydroxyapatite surfaces.¹¹²⁹

Anyway, the Fourier-transformed infrared (FTIR) spectra of some calcium orthophosphate-based composites and collagen films were measured and transformed into absorption spectra, using the Kramers-Kronig equation to demonstrate energy shifts of residues on the apatite/collagen interface. After comparing FTIR spectra of biocomposites and collagen films in detail, red shifts of the absorption bands for C-O bonds were observed in the spectra of the biocomposites. These red shifts were described as a decrease of bonding energies of C-O bonds and assumed to be caused by an interaction with Ca²⁺ ions located on the surfaces of apatite nano-sized crystals as shown in Figure 8.⁷⁴⁷ Another proof of a chemical interaction between apatite and collagen was also evaluated in FTIR spectra of CDHA/collagen biocomposites, in which a shift of the band corresponding to -COO^- stretching from 1,340 to 1,337 cm⁻¹ was observed.^708,709 More to the point, nucleation of apatite crystals onto collagen through a chemical interaction with carboxylate groups of collagen macromolecules has been reported in references 1130–1132.

Figure 8.

A schematic drawing of the relation between self-organization (directional deposition of HA on collagen) and interfacial interaction in biocomposites. Direction of interaction between HA and collagen is restricted by covalent bonding between COO and Ca(2) to maintain regular coordination number of 7. Reprinted from reference 747 with permission.

FTIR spectroscopy seems to be the major tool for studying a possible chemical bonding among the phases in calcium orthophosphate-based biocomposites and hybrid biomaterials.^{276,310,345,353,355,462,513,600,616,629,635,638,641,644,653,664,673,678,709,753,802,803,847,882,1133–1136} For example, the characteristic bands at 2,918, 2,850 and 1,472 cm⁻¹ for the hydrocarbon backbone of PE appeared to have zero shift in an HA/PE biocomposite. However, in the case of polyamide, several FTIR bands indicated that the polar groups shifted significantly: the bands at 3,304, 1,273 and 692 cm⁻¹ derived from stretching of N-H, stretching of C-N-H and vibrating of N-H moved to 3,306, 1,275 and 690 cm⁻¹, respectively, in the HA/polyamide biocomposites. Furthermore, both stretching (3,568 cm⁻¹) and vibrating (692 cm⁻¹) modes of hydroxide in HA moved to 3,570 and 690 cm⁻¹ in the HA/polyamide biocomposites, respectively, indicating the formation of hydrogen bonds. In addition, bands at 1,094 and 1,031 cm⁻¹ of PO₄ modes also shifted to 1,093 and 1,033 cm⁻¹ in the HA/polyamide biocomposite. That the bands shifted in a fingerprint area indicated that the hydroxide and orthophosphate on the surface of HA might interact with plentiful carboxyl and amino groups of polyamide through nucleophilic addition.²⁷⁶ Comparable conclusions were made for HA/PVA,⁶⁴¹ CDHA/alginate,⁷⁰⁹ ACP/PPF,⁵¹³ HA/maleic anhydride³⁵⁵ and β-TCP/PLLA⁴⁶² biocomposites, in which weak chemical bonds were considered to form between Ca²⁺ ions located on the HA, CDHA, ACP or β-TCP surface, respectively, and slightly polarized O atoms of C = O bonds in the surrounding bioorganic compounds. The data obtained suggest that crystallization of calcium orthophosphates in chitosan-containing solutions is substantially modulated by a chemical interaction of the components; apparently, a part of calcium is captured by chitosan and does not participate in the formation of the main mineral phase.¹¹³⁶ This type of the chemical interaction is shown schematically in Figure 9.⁷⁰⁹

Figure 9.

A schematic diagram of Ca²⁺ ion binding with alginate chains. Reprinted from reference 709 with permission.

Besides FTIR spectroscopy, other measurement techniques are also able to show some evidence of a chemical interaction among the phases in calcium orthophosphate-based biocomposites and hybrid biomaterials.^{345,462,635,638,641,1134–1138} For example, for nano-sized crystals of CDHA/alendronate such evidence was obtained by thermogravimetric analysis: DTG plots of the crystals appeared to be quite different from those obtained from mechanical mixtures of CDHA and calcium alendronate with similar compositions.¹¹³⁷ Analogous DTG results were obtained for nano-sized HA/PVA biocomposites.⁶⁴¹ In the case of biocomposites of nano-sized HA with polyamide, a hydrogen bonding among the phases was detected by a differential scanning calorimetry technique.⁶³⁵ Another example comprises application of dynamic mechanical analysis to investigate the softening mechanism of β-TCP/PLLA biocomposites.⁴⁶² As to biocomposites of nano-sized HA with PVAP, some indirect evidence of chemical bonding among the phases was found by X-ray diffraction and thermogravimetric analysis.³⁴⁵ A strong structural correlation between the orientation of FA crystallites and gelatin within the FA/gelatin composite spheres was discovered, indicating a substantial reorganization of the macromolecular matrix within the area of a growing aggregate.⁴⁴⁴ Recently, chemical interactions between HA and organic molecules have been elucidated using ab initio calculation methods.¹¹³⁹

By means of the X-ray photoelectronic spectroscopy (XPS) technique, binding energies of Ca, P and O atoms were found to vary between nano-sized HA (Ca: 350.5 and 345.5; O: 530.2; P: 132.5 eV) and nano-sized HA/konjac glucomannan/chitosan biocomposite (Ca: 352.1 and 347.4; O: 531.2; P: 133.4 eV).⁶⁵³ Further measurements by FTIR and X-ray diffraction revealed that nano-sized HA was mainly linked to konjac glucomannan and chitosan by hydrogen bonding among OH^- and PO₄^3- ions of HA and -C = O and -NH groups of konjac glucomannan and chitosan copolymer, and there was a stable interface formed among the three phases in the biocomposite. Meanwhile, coordinate bonding might be formed between Ca²⁺ and -NH. Stable interfaces have been formed among the three phases in a biocomposite.⁶⁵³ In HA/collagen biocomposites, a covalent bond formation between Ca²⁺ ions of HA and RCOO^- groups of collagen molecules was found by XPS.⁶⁰¹ Similar XPS observations were also made for several other calcium orthophosphate-based biocomposites and hybrid biomaterials.^629,664,673

The interaction and adhesion between calcium orthophosphate fillers and their respective matrices have significant effects on the properties of particulate-filled, reinforced materials, as these forces are essential to load transfer among the phases and thus to improving the mechanical performance of the biocomposites.³⁵³ However, for a substantial amount of the aforementioned formulations, the interaction among the phases is mechanical in nature. This is because the matrix often consists of compounds with no functional groups or unsaturated bonds that can form ionic complexes with the constituents of calcium orthophosphates. Obviously, less coupling exists between non-polar polymers and calcium orthophosphate ceramic particles. Therefore, polymers with functional groups pendant to the polymer backbone, which can act as sites for bridging to calcium orthophosphates, are more promising in this respect.⁵³

In order to improve the situation, various supplementary reagents are applied. If the primary effect of a processing additive is to increase the interaction between the phases, such additives can be regarded as coupling agents.¹¹⁴⁰ These agents establish chemical bridges between the matrix and the fillers, promoting adhesion among the phases. In many cases, their effect is not unique; they might, for example, also influence rheology of the composites.²⁸² In the case of calcium orthophosphates, a hexamethylene diisocyanate coupling agent was used to bind PEG/PBT (Polyactive^TM) block copolymers²⁹³ and other polymers¹¹³³ to HA filler particles. Thermogravimetric and infrared analysis demonstrated that the polymers were chemically bonded to the HA particles through the isocyanate groups, making it a suitable approach to improve adhesion.¹¹³³ Other researchers used glutaraldehyde as a cross-linked reagent.^{470,474,598,600,601,619,624,699,736,778,781,1141} Alternatively, the interfacial bonding among calcium orthophosphates and other components might be induced by silanes,^{242,268,269,293,406,640,1142–1145} zirconates,^{282,406,408,1146,1147} titanates,^282,406,1146 phosphoric acid,⁶⁴³ alkaline pretreatment,^877,880 polyacids^143,144,293 and some other chemicals. Furthermore, some polymers might be grafted onto the surface of calcium orthophosphate particles.⁶⁵⁶ Structural modifications of the polymeric matrices, for instance, with introduction of acrylic acid,^{245,268,269,293} have also proved to be effective. For example, application of polyacids as a bonding agent for HA/Polyactive^TM composites caused the surface modified HA particles to maintain better contact with polymers at the fracture and improved mechanical properties.^143,144,293 The use of titanate and zirconate coupling agents appeared to be very dependent on the molding technique employed.²⁸² Silane-coupled HA powders were tested before applying them as fillers in biodegradable composites.^1143–1145 This treatment allowed HA to withstand the attack of water without impairing overall bioactivity. Besides that, a chemically modified reinforcement phase-matrix interface was found to improve the mechanical properties of the biocomposites. The examples include chemically coupled HA/PE,^268,269 chemically formed HA/Ca poly(vinylphosphonate)³⁴⁹ and PLA/HA fibers.²³³ These biocomposites are able to consume a large amount of energy at the fracture.

The action of some coupling agents was found to combine two distinct mechanisms: (1) cross-linking of the polymeric matrix (valid for zirconate and titanate coupling agents) and (2) improvement of the interfacial interactions among the major phases of the biocomposites. This interfacial adhesion improvement appeared to be much dependent on the chemical nature (pH and type of metallic center) of the coupling agents.⁴⁰⁶ Several works claimed that silanes do interact with HA.^{242,268,269,1143–1145} It was shown that a silicon-containing interphase existed between HA and PE that promoted chemical adhesion between the HA particles and the polymer. A silane-coupling agent also facilitated penetration of PE into cavities of individual HA particles, which resulted in enhanced mechanical interlocking at the matrix-reinforcement interface.^268,269

Thus, the optimization of biocomposite properties by coupling agents is currently an important area of the research. The control and development of molecular-level associations of polymers with calcium orthophosphates is suggested to be significant for the resulting mechanical responses in biocomposites. It appears that a fundamental molecular understanding of the interfacial behavior in biocomposites is an area not sufficiently addressed in the literature. Various experimental characterization techniques using electron microscopy, vibrational spectroscopy, X-ray diffraction, scanning probe microscopy and others are used routinely to characterize these materials beyond mechanical property characterization. In addition, atomic scale models for simulating phase interaction and predicting responses in the novel material systems, where nanostructures and nano-interfaces are included, are important to understand and predict load deformation behavior.¹⁷⁹

In addition to the aforementioned, the surface of calcium orthophosphates might be modified as well.^{144,509,510,656,1147–1154} An interesting approach for HA surface modification was described by Lee et al.¹¹⁵⁴ First, in situ synthesis of surface thiol-functionalized HA (HA-SH) was realized by adding 3-mercaptopropionic acid during hydrothermal synthesis of HA (Fig. 10A). This was followed by grafting polymerization of ethylene glycol methacrylate phosphate by radical chain transfer, generating the sulfur-centered radicals on the HA surfaces (Fig. 10B), which initiated the surface grafting polymerization of ethylene glycol methacrylate phosphate (Fig. 10C).¹¹⁵⁴ Other examples might be found in the literature.^{144,509,510,656,1147–1153} In general, the purpose of surface modifying is not only to guarantee the even distribution of calcium orthophosphate particles at a high loading level in the matrix, but also to prevent or delay the debonding process of calcium orthophosphate particles from the matrix. Obviously, all surface modifiers must satisfy several biomedical requirements, such as no toxicity, good biocompatibility and no changes in the biological or physicochemical properties of the fillers.

Figure 10.

Surface modification of HA particles by grafting polymerization according to Lee et al.¹¹⁵⁴ (A) surface thiol functionalized HA, (B) sulfur-centered radical on HA surface, (C) surface grafting polymerization of ethylene glycol methacrylate phosphate. Reprinted from reference 99 with permission.

Addition of adhesion-promoting agents might be an alternative to improve the interaction between the fillers and the matrix. For example, Morita et al. incorporated 4-methacryloyloxyethyl trimellitate anhydride to promote adhesion of the polymer to HA.¹¹⁵⁵ In another study, phosphoric ester was added to the liquid component of the formulation.¹¹⁵⁶ Both the strength and the affinity index of biocomposites were found to increase, probably due to the effects of co-polymerization.

Possible interactions between BCP and HPMC have been investigated in IBS composites.^900,901,1157 After mixing, there was a decrease in the mean diameter of BCP granules, and this influenced the viscosity of the paste. Dissolution of grain boundaries of β-TCP crystals and precipitation of CDHA on the HA crystal surface were found during the interaction. Both phenomena were responsible for the observed granulometric changes;^900,901 however, within the sensitivity of the employed measurement techniques, no chemical bonding between BCP and HPMC was detected.¹¹⁵⁷

A coprecipitation technique was used to prepare CDHA/chitosan biocomposites.⁸¹¹ Growth of CDHA crystals was inhibited by organic acids with more than two carboxyl groups, which strongly bind to CDHA surfaces via COO-Ca bonds. Transmission electron microscopy images revealed that CDHA formed elliptic aggregates with chemical interactions (probably coordination bond) between Ca on its surface and amino groups of chitosan; the nano-sized crystals of CDHA were found to align along the chitosan molecules, with the amino groups working as the nucleation sites.⁸¹¹ Formation of calcium cross-linked polymer carboxylate salts was suggested during setting of calcium orthophosphate cement (TTCP + DCPA)/polyphosphazane biocomposites; a chemical involvement of the polymer in the cement setting was determined based on the results of pH monitoring.^558-560

A chemical bond between the phases was presumed in PCL/ HA composites prepared by the grafting technique;⁴²⁰ unfortunately, no strong experimental evidences were provided. In another study, CDHA/poly(α-hydroxyester) composites were prepared by a low temperature chemical route.³⁹³ In that study, pre-composite structures were prepared by combining α-TCP with PLA, PLGA and copolymers thereof. The final biocomposite was achieved by in situ hydrolysis of α-TCP to CDHA performed at 56°C either in solvent cast or pressed pre-composites. That transformation occurred without any chemical reaction between the polymer and calcium orthophosphates, as determined by FTIR spectroscopy.³⁹³

In nearly every study on HA/carbon nanotubes biocomposites, the nanotubes were functionalized before combining them with HA. Most researchers did this by oxidation,^303-307 although noncovalent functionalizing with sodium dodecylsulfate³⁰⁷ and coating the nanotubes by a polymer¹¹⁵⁸ before combining them with HA were also reported. Several studies by transmission electron microscopy revealed evidence that the functionalization enhanced interaction between carbon nanotubes and HA.^306,307,1159

For calcium orthophosphate-based biocomposites able to sustain high-temperature sintering (valid for the formulations consisting of inorganic components only), an interdiffusion of chemical elements might take place among the phases. Such an effect was detected by energy-dispersive X-ray spectroscopy in HA/TiO₂ biocomposite particles with partial formation of calcium titanates; this process was found to be favorable to enhancing the cohesive strength of particles in the composite coating.⁹⁹⁷ A similar high-temperature interaction between HA and zirconia^911,940 as well as between HA and Ti^{661,1030,1032–1034} was also detected. Namely, lower Ti content composites sintered at 1,200°C showed main crystalline phases as CaTiO₃, CaO and Ti_xP_y, while an increase in Ti content to 50 vol.% revealed Ti₂O and residual α-Ti as additional phases. Thus, the chemical reactions between HA and Ti were expressed by the following unbalanced illustrative equation (Eqn. 1):¹⁰³²

Besides, partial decomposition of HA and formation of different calcium aluminates were detected in HA/Al₂O₃ biocomposites after sintering at 1,200–1,300°C. This has been attributed to the diffusion of Ca²⁺ from HA into the alumina matrix, and the depletion of Ca²⁺ from HA leads to the decomposition of HA into β-TCP.^968,974-976 Presumably, all these processes influence the mechanical strength of the biocomposites.

Bioactivity and Biodegradation of Calcium Orthophosphate-Based Formulations

The continuous degradation of an implant causes a gradual load transfer to the healing tissue, preventing stress shielding atrophy, and stimulates the healing and remodeling of bones. Some requirements must be fulfilled by the ideal prosthetic biodegradable materials, such as biocompatibility, adequate initial strength and stiffness, retention of mechanical properties long enough to assure its biofunctionality and the non-toxicity of the degradation by-products.¹⁷⁸ Generally speaking, bioactivity (i.e., ability of bonding to bones) of biologically relevant calcium orthophosphates reinforced by other materials is usually lower than that of pure calcium orthophosphates.^30,31,1160

In general, both bioactivity and biodegradability of any biocomposite and/or hybrid biomaterial are determined by the same properties of the constituents. Both processes are very multi-factorial, because during implantation, the surface of any graft comes into contact with biological fluids and, shortly afterwards, is colonized by cells. Much more biology than chemistry and material science together is involved in these very complex processes, and many specific details still remain unknown. To simplify the task, the biodegradability of the biologically relevant calcium orthophosphates might be described by a chemical dissolution in slightly acidic media (calcium orthophosphates are almost insoluble in alkaline solutions^111-117), which, in the case of CDHA, might be described as a sequence of four successive chemical equations (Eqns. 2–5):^{519,1161,1162}

Biodegradability of polymers generally depends on the following factors: (1) chemical stability of the polymer backbone, (2) hydrophobicity of the monomer, (3) morphology of the polymer, (4) initial molecular weight, (5) fabrication processes, (6) geometry of the implant and (7) properties of the scaffold, such as porosity and pore diameter.³²⁸ A summary on degradation of PLA and PGA as well as that of SEVA-C is available in the literature (reviewed in ref. 178 p. 798 and p. 803, respectively), where the interested readers are referred. Biodegradation of HA/PLLA and CDHA/PLLA biocomposite rods in subcutis and medullary cavities of rabbits were investigated mechanically and histologically; the degradation was found to be faster when using uncalcinated CDHA instead of calcinated HA.¹¹⁶³ In a more detailed study, new bone formation was detected at 2 weeks after implantation, especially for formulations with a high HA content.¹¹⁶⁴ More to the point, direct contact between bones and these composites without intervening fibrous tissue was detected in this case.^1164,1165 Both SEVA-C and SEVA-C/ HA biocomposites were found to exhibit non-cytotoxic behavior,^1166,1167 inducing a satisfactory tissue response when implanted as shown by in vivo studies.¹¹⁶⁷ Furthermore, SEVA-C/HA biocomposites induce a positive response in osteoblast-like cells for what concerns cell adhesion and proliferation.¹¹⁶⁶ An in vivo study on biodegradation of microspheres [PLGA, gelatin and poly(trimethylene carbonate) were used]/calcium orthophosphate cement biocomposites revealed that they exhibited microsphere degradation after 12 weeks of subcutaneous implantation, which was accompanied by a decrease in compression strength.¹¹⁶⁸ Interestingly, though, the amount of calcium orthophosphates in biocomposites was found to have a greater effect on the early stages of osteoblast behavior (cell attachment and proliferation) rather than the immediate and late stages (proliferation and differentiation).¹¹⁶⁹

Both in vitro (the samples were immersed into 1% trypsin/phosphate-buffered saline solution at 37°C) and in vivo (implantation of samples into the posterolateral lumbar spine of rabbits) biodegradation have been investigated for nano-sized HA/collagen/PLA biocomposites.⁶¹⁰ The results demonstrated that weight loss increased continuously in vitro, with a reduction in mass of ~20% after 4 weeks. During the experimental period in vitro, a relative rate of reduction of the three components in this material was shown to differ greatly: collagen decreased the fastest, from 40% weight to ~20% in the composite; HA content increased from 45 to ~60%; PLA changed little. In vivo, the collagen/HA ratio appeared to be slightly higher near the transverse process than in the central part of the intertransverse process.⁶¹⁰ Hasegawa et al.¹¹⁷⁰ performed an in vivo study, spanning a period of 5–7 y, on high-strength HA/PLLA biocomposite rods for the internal fixation of bone fractures. In that work, both uncalcined CDHA and calcined HA were used as reinforcing phases in a PLLA matrix. Those composites were implanted in the femur of 25 rabbits. It was found that the implanted materials were resorbed after 6 y of implantation. The presence of remodeled bone and trabecular bone bonding was the significant outcome. These data clearly demonstrate the biodegradation independence of various components of biocomposites.

Some Challenges and Critical Issues

The scientific information summarized in this review represents the recent developments of calcium orthophosphate-based biocomposites and hybrid biomaterials from a variety of approaches, starting from conventional ones to tissue engineering. Such formulations combined with osteoconductive or osteoinductive factors and/or osteogenic cells have gained much interest as a new and versatile class of biomaterials and are perceived to be beneficial in many aspects as bone grafts.^36,1171 However, current applications of these biomaterials in medicine and surgery are still remarkably less than might be expected. In many biomedical applications, research and testing of such formulations have been introduced and highly developed, but only in a very few cases have industrial production and commercial distribution of medical devices partially or entirely made of biocomposites been started. The medical application of biocomposites and hybrid biomaterials requires a better understanding of the objectives and limitations involved. Recently, the main critical issues have been summarized as follows:²⁶⁵

• There are not enough reliable experimental and clinical data supporting the long-term performance of biocomposites with respect to monolithic traditional materials.

• The design of biocomposites and hybrid biomaterials is far more complex than that of conventional monolithic materials because of the large number of additional design variables that must be considered.

• The available fabrication methods may limit the possible reinforcement configurations, may be time consuming, expensive and may require special cleaning and sterilization processes as well as highly skilled personnel.

• There are no satisfactory standards yet for biocompatibility testing of the biocomposite implants, because the ways in which the different components of any biocomposite interact with living tissues are not completely understood.

• There are no adequate standards for the assessment of biocomposite fatigue performance, because the fatigue behavior of such materials is far more complex and difficult to predict than that of traditional materials.²⁶⁵

On the other hand, in spite of an enormous progress in biocomposite processing, to achieve the desired characteristics, researchers still need to develop more advanced technologies to fabricate a bone-resembling hierarchical organization over several length scales. Development of novel grafting materials depends on the progress of research into the structure of natural bones. The key issues are not only to understand the fundamentals of biomineralization but also to translate such knowledge into practical synthetic pathways to produce better bone grafts. Unfortunately, when it comes to the fabrication of biocomposites mimicing natural bones, from the nanometer to the micrometer dimensions, there are many key issues, including control of morphology, incorporation of foreign ions, interaction with biomolecules and assembly of the organic and inorganic phases, which are still not well understood. A processing gap between the lower-level building units and the higher-order architecture could severely limit the practical application of current calcium orthophosphate-based biocomposites and hybrid biomaterials. Therefore, further substantial research efforts have been outlined to address the following key challenges:^36,41

• Optimization of biocomposite processing conditions.

• Optimization of interfacial bonding and strength equivalent to natural bone.

• Optimization of the surface properties and pore size to maximize bone growth.

• Maintaining the adequate volume of the construct in vivo to allow bone formation to take place.

• Withstanding the load-bearing conditions.

• Matching the bioresorbability of the grafts and their biomechanical properties while forming new bone.

• Understanding the molecular mechanisms by which the cells and the biocomposite matrix interact with each other in vivo to promote bone regeneration.

• Supporting angiogenesis and vascularization for the growth of healthy bone cells and subsequent tissue formation and remodeling.^36,41

The aforementioned critical issues have to be solved before a widespread commercial use of calcium orthophosphate-based biocomposites and hybrid biomaterials can be made in surgery and medicine.

Conclusions

All types of calcified tissues of humans and mammals appear to possess a complex hierarchical biocomposite structure. Their mechanical properties are outstanding (considering the weak constituents from which they are assembled) and far beyond those, that can be achieved using the same synthetic materials with present technologies. This is because biological organisms produce biocomposites that are organized in terms of both composition and structure, containing both brittle calcium orthophosphates and ductile bioorganic components in very complex structures, hierarchically organized at the nano-, micro- and meso levels. Additionally, the calcified tissues are always multifunctional. For example, bone provides structural support for the body plus blood cell formation. The third defining characteristic of biological systems, in contrast with current synthetic systems, is their self-healing ability, which is nearly universal in nature. These complex structures, which have risen from millions of years of evolution, inspire materials scientists in the design of novel biomaterials.¹¹⁷²

Obviously, no single-phase biomaterial is able to provide all the essential features of bones and/or other calcified tissues, and therefore, there is a great need to engineer multi-phase biomaterials (biocomposites) with a structure and composition mimicking those of natural bones. The studies summarized in this review have shown that the proper combination of a ductile matrix with a brittle, hard and bioactive calcium orthophosphate filler offers many advantages for biomedical applications. Namely, the desirable properties of some components can compensate for a poor mechanical behavior of calcium orthophosphate bioceramics, while, in turn, the desirable bioactive properties of calcium orthophosphates improve those of other phases, thus expanding the possible application of each material within the body.¹⁰² However, the reviewed literature clearly indicates that, among possible types of calcium orthophosphate-based biocomposites and hybrid biomaterials, only simple, complex and graded ones, as well as fibrous, laminar and particulate ones (see classification types of the composites in the “General Information on Composites and Biocomposites” section) have been investigated. Presumably, future progress in this subject will require concentrating efforts on elaboration and development of both hierarchical and hybrid biocomposites. Furthermore, following the modern tendency of tissue engineering, a novel generation of calcium orthophosphate-based biocomposites and hybrid biomaterials should also contain a living biological part.

To conclude, the future of the calcium orthophosphate-based biocomposites and hybrid biomaterials is now directly dependent on the formation of multidisciplinary teams composed of experts but, primarily, experts ready to work in close collaboration with others and thus be able to deal efficiently with the complexity of the human organism. The physical chemistries of solids, solid surfaces, polymer dispersion and solutions as well as material-cell interactions are among the phenomena to be tackled. Furthermore, much work remains to be done on the long way from laboratory to clinic, and success depends on the effective cooperation of clinicians, chemists, biologists, bioengineers and materials scientists.

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However, there are some reports about a lack of TCP biodegradation after implantation in calvarial defects.

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Glossary

Abbreviations:

A-W

apatite-wollastonite

BMP

bone morphogenetic protein

BSA

bovine serum albumin

EVOH

a copolymer of ethylene and vinyl alcohol

HDPE

high-density polyethylene

HIPS

high impact polystyrene

HPMC

hydroxypropylmethylcellulose

IBS

injectable bone substitute

PAA

polyacrylic acid

PBT

polybutyleneterephthalate

PCL

poly(∑-caprolactone)

PDLLA

poly(D,L-lactic acid)

PE

polyethylene

PEEK

polyetheretherketone

PEG

polyethylene glycol

PGA

polyglycolic acid

PHB

polyhydroxybutyrate

PHBHV

poly(hydroxybutyrate-co-hydroxyvalerate)

PHEMA

polyhydroxyethyl methacrylate PHV, polyhydroxyvalerate

PLA

polylactic acid

PLGA

poly(lactic-co-glycolic) acid

PLGC

co-polyester lactide-co-glycolide-co-∑-caprolactone

PLLA

poly(L-lactic acid)

PMMA

polymethylmethacrylate

PP

polypropylene

PPF

poly(propylene-co-fumarate)

PS

polysulfone

PSZ

partially stabilized zirconia

PTFE

polytetrafluoroethylene

PVA

polyvinyl alcohol

PVAP

polyvinyl alcohol phosphate

SEVA-C

a blend of EVOH with starch

UHMWPE

ultrahigh molecular weight polyethylene